León A. Bravo

Freezing resistance varies within the growing season and with elevation in high-Andean species of central Chile.

Predicted increases in the length of the growing season as a result of climate change may more frequently expose high-elevation plants to severe frosts. Understanding the ability of these species to resist frosts during the growing season is essential for predicting how species may respond to changes in temperature regimes. Here, we assessed the freezing resistance of 24 species from the central Chilean Andes by determining their low temperature damage (LT(50)), ice nucleation temperature (NT), freezing point (FP) and freezing resistance mechanism (i.e. avoidance or tolerance). The Andean species were found to resist frosts from -8.2 to -19.5 degrees C during the growing season, and freezing tolerance was the most common resistance mechanism. Freezing resistance (LT(50)) varied within the growing season, decreasing towards the end of this period in most of the studied species. However, the FP showed the opposite trend. LT(50) increased with elevation, whilst FP was lower in plants from lower elevations, especially late in the growing season. Andean species have the potential to withstand severe freezing conditions during the growing season, and the aridity of this high-elevation environment seems to play an important role in determining this high freezing resistance.

Calcium Interacts with Antifreeze Proteins and Chitinase from Cold-Acclimated Winter Rye

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca2+-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca2+-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca2+-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.

Ecotypic Differentiation in Morphology and Cold Resistance in Populations of Colobanthus quitensis (Caryophyllaceae) from the Andes of Central Chile and the Maritime Antarctic

Abstract Colobanthus quitensis is one of only two vascular plant species that occur in the Antarctic islands. We evaluated morphological and physiological traits and survival after freezing in individuals of C. quitensis collected in two populations: one from the Andes of central Chile and the other from the maritime Antarctic. We addressed whether these populations are ecotypes in terms of shoot morphology and physiology, and cold resistance. We also evaluated genetic differentiation at the internal transcribed spacer (ITS) regions. Under controlled growing conditions, Andean plants had longer and narrower leaves and longer peduncles. However, shoot biomass was similar in both populations. There was a 1.17% sequence divergence of ITS regions between populations. Survival to freezing at temperatures from 0° to −16°C was not different in nonacclimated plants. After cold-acclimation at 4°C, freezing tolerance was greater in Antarctic plants. Both Andean and Antarctic plants increased soluble sugars with increasing acclimation time, but it did not differ between populations. Shoot water content decreased with acclimation time in both populations, but Antarctic plants maintained slightly higher water contents after cold exposure. Shoot growth did not differ during acclimation. The studied populations of C. quitensis represent different morphological and cold resistant ecotypes despite their genetic similarity. However, the mechanistic basis of the higher survival to freezing of Antarctic plants was not elucidated.

The role of photochemical quenching and antioxidants in photoprotection of Deschampsia antarctica

Deschampsia antarctica Desv. (Poaceae) is the only grass that grows in the maritime Antarctic. Constant low temperatures and episodes of high light are typical conditions during the growing season at this latitude. These factors enhance the formation of active oxygen species and may cause photoinhibition. Therefore, an efficient mechanism of energy dissipation and / or scavenging of reactive oxygen species (ROS) would contribute to survival in this harsh environment. In this paper, non-acclimated and cold-acclimated D. antarctica were subjected to high light and / or low temperature for 24 h. The contribution of non-photochemical dissipation of excitation light energy and the activities of detoxifying enzymes in the development of resistance to chilling induced photoinhibition were studied by monitoring PSII fluorescence, total soluble antioxidants, and pigments contents and measuring variations in activity of superoxide dismutase (SOD; EC 1.15.1.1), ascorbate peroxidase (APX; EC 1.11.1.11), and glutathione reductase (GR; EC 1.6.4.2). The photochemical efficiency of PSII, measured as Fv / F m, and the yield of PSII electron transport (ΦPSII) both decreased under high light and low temperatures. In contrast, photochemical quenching (qP) in both non-acclimated and cold-acclimated plants remained relatively constant (approximately 0.8) in high-light-treated plants. Unexpectedly, qP was lower (0.55) in cold-acclimated plants exposed to 4°C and low light intensity. Activity of SOD in cold-acclimated plants treated with high light at low temperature showed a sharp peak 2-4 h after the beginning of the experiment. In cold-acclimated plants APX remained high with all treatments. Activity of GR decreased in cold-acclimated plants. Compared with other plants, D. antarctica exhibited high levels of SOD and APX activity. Pigment analyses show that the xanthophyll cycle is operative in this plant. We propose that photochemical quenching and particularly the high level of antioxidants help D. antarctica to resist photoinhibitory conditions. The relatively high antioxidant capacity of D. antarctica may be a determinant for its survival in the harsh Antarctic environment.

Cold-acclimation limits low temperature induced photoinhibition by promoting a higher photochemical quantum yield and a more effective PSII restoration in darkness in the Antarctic rather than the Andean ecotype of Colobanthus quitensis Kunt Bartl (Cariophyllaceae)

BackgroundEcotypes of Colobanthus quitensis Kunt Bartl (Cariophyllaceae) from Andes Mountains and Maritime Antarctic grow under contrasting photoinhibitory conditions, reaching differential cold tolerance upon cold acclimation. Photoinhibition depends on the extent of photodamage and recovery capability. We propose that cold acclimation increases resistance to low-temperature-induced photoinhibition, limiting photodamage and promoting recovery under cold. Therefore, the Antarctic ecotype (cold hardiest) should be less photoinhibited and have better recovery from low-temperature-induced photoinhibition than the Andean ecotype. Both ecotypes were exposed to cold induced photoinhibitory treatment (PhT). Photoinhibition and recovery of photosystem II (PSII) was followed by fluorescence, CO2 exchange, and immunoblotting analyses.ResultsThe same reduction (25%) in maximum PSII efficiency (Fv/Fm) was observed in both cold-acclimated (CA) and non-acclimated (NA) plants under PhT. A full recovery was observed in CA plants of both ecotypes under dark conditions, but CA Antarctic plants recover faster than the Andean ecotype.Under PhT, CA plants maintain their quantum yield of PSII, while NA plants reduced it strongly (50% and 73% for Andean and Antarctic plants respectively). Cold acclimation induced the maintenance of PsaA and Cyt b6/f and reduced a 41% the excitation pressure in Antarctic plants, exhibiting the lowest level under PhT. xCold acclimation decreased significantly NPQs in both ecotypes, and reduced chlorophylls and D1 degradation in Andean plants under PhT.NA and CA plants were able to fully restore their normal photosynthesis, while CA Antarctic plants reached 50% higher photosynthetic rates after recovery, which was associated to electron fluxes maintenance under photoinhibitory conditions.ConclusionsCold acclimation has a greater importance on the recovery process than on limiting photodamage. Cold acclimation determined the kinetic and extent of recovery process under darkness in both C. quitensis ecotypes. The greater recovery of PSII at low temperature in the Antarctic ecotype was related with its ability to maintain PsaA, Cyt b6/f and D1 protein after photoinhibitory conditions. This is probably due to either a higher stability of these polypeptides or to the maintenance of their turnover upon cold acclimation. In both cases, it is associated to the maintenance of electron drainage from the intersystem pool, which maintains QA more oxidized and may allow the synthesis of ATP and NADPH necessaries for the regeneration of ribulose 1,5-bisphosphate in the Calvin Cycle. This could be a key factor for C. quitensis success under the harsh conditions and the short growing period in the Maritime Antarctic.

Changes in morpho-physiological attributes of Eucalyptus globulus plants in response to different drought hardening treatments

Morpho-physiological attributes exhibited in response to drought hardening at the end of the growing season of Eucalyptus globulus Labill under nursery conditions were studied to evaluate the effect of three drought hardening treatments in morpho-physiological traits used as suitable indicators of drought hardiness, such as, plant growth, root growth potential, plant water relationships and survival. Freezing resistance of drought hardened plants was also studied in order to evaluate cross hardening effects in cuttings of Eucalyptus globulus Labill. Drought hardening consisted in induced water stress by watering restriction, until plant stem xylem water potentials (Ψ pd ) reached to-0.2, -1.3 and -2.4 MPa. Two water stress-rewatering cycles were applied during 54 days of treatment. The hardening treatments caused a significant reduction in plant height, leaf area, specific leaf area, plant, leaf, stem and root biomass. However, stem diameter was not affected. Root growth potential increased with the exposure to moderate water stress (-1.3 MPa). Drought hardening treatments have not effect on water relationship parameters such as saturation osmotic potential (Ψπ sat ), volumetric module of elasticity (e), relative water content (RWC tlp ) and osmotic potential (Ψπ tlp ) at the turgor loss point. Only 1.7% and 6% of dehydrated dead plants were observed on treatments at -1.3 and -2.4 MPa respectively. Finally, the freezing damage index of leaves (LT 50 ) was not significantly affected by drought hardening treatments. Furthermore, a reduction of 1.1oC of supercooling capacity was observed at -2.4 MPa. As a conclusion, drought hardening is an important step of plants production programs during the final phase of nursery, because changes in morphological attributes caused by exposure to moderate drought, enable the plants to maintain the balance between transpiration and absorption areas and increase the capacity of plants to generate new roots.

Identification and characterization of three novel cold acclimation-responsive genes from the extremophile hair grass Deschampsia antarctica Desv.

Deschampsia antarctica Desv. is the only monocot that thrives in the harsh conditions of the Antarctic Peninsula and represents an invaluable resource for the identification of genes associated with freezing tolerance. In order to identify genes regulated by low temperature, we have initiated a detailed analysis of its gene expression. Preliminary 2-D gels of in vivo-labeled leaf proteins showed qualitative and quantitative differences between cold-acclimated and non-acclimated plants, suggesting differential gene expression. Similarly, cold-acclimation-related transcripts were screened by a differential display method. Of the 38 cDNAs initially identified, three cDNA clones were characterized for their protein encoding, expression pattern, response to several stresses, and for their tissue-specific expression. Northern blot analysis of DaGrx, DaRub1, and DaPyk1 encoding a glutaredoxin, a related-to-ubiquitin protein, and a pyruvate kinase-like protein, respectively, showed a distinct regulation pattern during the cold-acclimation process, and in some cases, their cold response seemed to be tissue specific. All three transcripts seem to be responsive to water stress as their levels were up-regulated with polyethyleneglycol treatment. DaRUB1 and DaPyk1 expression was up-regulated in leaf and crown, but down-regulated in roots from cold-acclimated plants. The significance of these results during the cold-acclimation process will be discussed.

Photosynthetic and leaf anatomical characteristics of Castanea sativa: a comparison between in vitro and nursery plants

The anatomic and functional leaf characteristics related to photosynthetic performance of Castanea sativa growing in vitro and in nursery were compared. The irradiance saturated photosynthesis in in vitro grown plantlets was significantly lower compared to nursery plants (65 vs. 722 μmol m−2 s−1). The maximum photosynthetic rate (PNmax) was 4.0 and 10.0 μmol(CO2) m−2 s−1 in in vitro microshoots and nursery plant leaves, respectively. Carboxylation efficiency (CE) and electron transport rate (ETR) were three-folds higher in nursery plants than in microshoots. The nonphotochemical quenching (NPQ) was saturated at 80 μmol m−2 s−1 in microshoots suggesting limited photoprotection by thermal dissipation. The microshoots had wide open, spherical stomata and higher stomatal density than nursery plants and they had almost no epicuticular wax. Consequently, the microshoots had high stomatal conductance and high transpiration rate. These anatomic and functional leaf characteristics are likely major causes of the low survival rates of plantlets after ex vitro transfer.

Light energy management in micropropagated plants of Castanea sativa, effects of photoinhibition.

The limited development of photoprotective mechanisms, specifically heat dissipation capacity, found in micropropagated plants may be the result of low xanthophyll cycle pigment content and reduced de-epoxidation capacity making them highly susceptible to photodamage. The effects of gradual or sudden increase of light on Castanea sativa in vitro cultured and during their ex vitro transference was evaluated. The results were compared with those determined in nursery-grown plants. In vitro plants responded poorly to gradual increase in irradiance, exhibiting a low electron transport rate (ETR) agreeing with low non-photochemical quenching (NPQ) and a limited de-epoxidation capacity, not synthesizing detectable amounts of zeaxanthin (Z). Regarding a sudden increase in light (photoinhibition treatment, PhT); post-PhT as in vitro as well nursery plants showed a significant decrease in their maximal efficiency of PSII (F(v)/F(m)), but in vitro the decrease was very drastic (around 0.2) different from that observed in nursery (around 0.69). In vitro, NPQ was mainly determined by the slow relaxing component, NPQ(s) (80.8%), concomitant with a pronounced decrease of D1 protein post-PhT, and a lack of de-epoxidation capacity. During ex vitro transfer, PhT lead to death of some plants, specifically during root induction. The photoprotective mechanisms were activated over time in ex vitro conditions, indicating that micropropagated Castanea sativa display a potential for light acclimation, adjusting their photosynthetic apparatus to the ambient growth irradiance. Understanding the mechanisms that micropropagated plants deployed and how they face high light intensity events, will allow us to search for strategies to improve performance to possible light fluctuations that normally occur in ex vitro conditions during plant acclimation.

Light regulation of sucrose-phosphate synthase activity in the freezing-tolerant grass Deschampsia antarctica

Deschampsia antarctica, a freezing-tolerant grass that has colonized the Maritime Antarctic, has an unusually high content of sucrose (Suc) in leaves, reaching up to 36% of dry weight. Suc accumulation has often been linked with increased activity of sucrose phosphate synthase (SPS; EC: 2.4.1.1.14). SPS, a key enzyme in sucrose biosynthesis, is controlled by an intricate hierarchy of regulatory mechanisms including allosteric modulators, reversible covalent modification in response to illumination, and transcriptional regulation. We hypothesized that during long day conditions in the Antarctic summer D. antarctica can maintain high SPS activity longer by indirect light regulation, thereby leading to a high sucrose accumulation in the leaves. The objectives of this study were to investigate a possible indirect light regulation of SPS activity and the effect of cold and day length on transcriptional and protein level of SPS in D. antarctica. Although SPS activity did not display an endogenous rhythm of activity in continuous light, activation of SPS at the end of the dark period was observed in D. antarctica. This activation of SPS is possibly controlled by covalent modification, because it was inhibited by okadaic acid while the SPS protein level did not significantly change. The highest SPS activity increase was observed after 21 days of cold-acclimation under long day conditions. This increased activity was not related to an increase in SPS gene expression or protein content. High SPS activity in cold long days leading to hyper accumulation of Suc appears to be among the features that permit D. antarctica to survive in the harsh Antarctic conditions.