Cryoprotective activity of a cold-induced dehydrin purified from barley

Dehydrins are a family of proteins associated with cell dehydration. Drought, salinity, and high and low temperature may cause water loss from cells. Cold‐induced dehydrins have been reported in several species. P‐80 is a cold‐induced 80 kDa dehydrin in barley. This protein has the same apparent molecular mass as Dhn5, previously described for barley cv Himalaya. P‐80 was localized in the vicinity of vascular cylinders and in the epidermis of leaves and stems. Both tissues have been reported to be sites of early ice nucleation during controlled freezing. The present authors have proposed that this protein cryoprotects macromolecules and frost‐sensitive structures. In the present study, P‐80 and Dhn5 were purified with the purposes of demonstrating their cryoprotective activity in vitro, and comparing both proteins. More than 95% purity was obtained combining heat treatment, cationic exchange chromatography, preparative denaturant electrophoresis and band electroelution. Western blots showed that P‐80 was the major cold‐induced dehydrin in the cultivars examined in the present study. There was a major band of mRNA that showed expression kinetics consistent with P‐80 accumulation. The RT‐PCR picked one major band when using Dhn5‐specific primers in four cold‐acclimated barley cultivars. Both proteins have a similar amino acid composition, with differences in Arg, Asn + Asp, Glu + Gln, His, and Lys. The analysis of proteolytic fragments of Dhn5 and P‐80 by reverse phase chromatography showed a similar pattern. Furthermore, both proteins were able to cryoprotect lactate dehydrogenase (LDH, EC 1.1.1.27) against freeze/thaw inactivation, showing a similar shape dependence on concentration and almost the same protein dosage that renders 50% of cryoprotection (PD₅₀). Thus, P‐80 and Dhn‐5 share more similarities than expected for two different proteins. Their identities, though, remain to be firmly established. Further research is necessary to establish if the observed in vitro cryoprotective activity of these dehydrins is important for cryoprotection in vivo. The association of cryoprotective activity with K repeats of dehydrins is discussed.     

Ecophysiology of Antarctic vascular plants

Most of the ice and snow-free land in the Antarctic summer is found along the Antarctic Peninsula and adjacent islands and coastal areas of the continent. This is the area where most of the Antarctic vegetation is found. Mean air temperature tends to be above zero during the summer in parts of the Maritime Antarctic. The most commonly found photosynthetic organisms in the Maritime Antarctic and continental edge are lichens (around 380 species) and bryophytes (130 species). Only two vascular plants, Deschampsia antarctica Desv. and Colobanthus quitensis (Kunth) Bartl., have been able to colonize some of the coastal areas. This low species diversity, compared with the Arctic, may be due to permanent low temperature and isolation from continental sources of propagules. The existence of these plants in such a permanent harsh environment makes them of particular interest for the study of adaptations to cold environments and mechanisms of cold resistance in plants. Among these adaptations are high freezing resistance, high resistance to light stress and high photosynthetic capacity at low temperature. In this paper, the ecophysiology of the two vascular plants is reviewed, including habitat characteristics, photosynthetic properties, cold resistance, and biochemical adaptations to cold.     

Cloning and characterisation of the Anisakis simplex allergen Ani s 4 as a cysteine-protease inhibitor

Anisakis simplex is a nematode that can parasitise humans who eat raw or undercooked fish containing live L3s. Larvae invading the gastrointestinal mucosa excrete/secrete proteins implicated in the pathogenesis of anisakiasis that can induce IgE mediated symptoms. Misdiagnosis of anisakiasis, due to cross-reactivity, makes it necessary to develop new diagnostic tools. Recombinant allergens have proved to be useful for diagnosis of other parasitoses. Among the Anisakis allergens, Ani s 4 was considered to be a good potential diagnostic protein because of its heat resistance and its importance in the clinical history of sensitised patients. Therefore, the objective of this study was to clone and characterise the cDNA encoding this allergen. The Ani s 4 mRNA sequence was obtained using a PCR-based strategy. The Ani s 4 amino acid sequence contained the characteristic domains of cystatins. Mature recombinant Ani s 4 was expressed in a bacterial system as a His-tagged soluble protein. The recombinant Ani s 4 inhibited the cleavage of a peptide substrate by papain with a Ki value of 20.6 nM. Immunobloting, ELISA, a commercial fluorescence-enzyme-immunoassay and a basophil activation test were used to study the allergenic properties of rAni s 4, demonstrating that the recombinant allergen contained the same IgE epitopes as the native Ani s 4, and that it was a biologically active allergen since it activated basophils from patients with allergy to A. simplex in a specific concentration-dependent manner. Ani s 4 was localised by immunohistochemical methods, using a polyclonal anti-Ani s 4 anti-serum, in both the secretory gland and the basal layer of the cuticle of A. simplex L3. In conclusion, we believe that Ani s 4 is the first nematode cystatin that is a human allergen. The resulting rAni s 4 retains all allergenic properties of the natural allergen, and can therefore be used in immunodiagnosis of human anisakiasis.     

Is electron transport to oxygen an important mechanism in photoprotection? Contrasting responses from Antarctic vascular plants

Photoreduction of oxygen by the photosynthetic electron transport chain has been suggested to be an important process in protecting leaves from excess light under conditions of stress; however, there is little evidence that this process occurs significantly except when plants are exposed to conditions outside their normal tolerance range. We have examined the oxygen dependency of photosynthetic electron transport in the two vascular plants found growing in Antarctica – Colobanthus quitensis and Deschampsia antarctica. Photosynthetic electron transport in C. quitensis is insensitive to changes in oxygen concentration under non-photorespiratory conditions, indicating that electron transport to oxygen is negligible; however, it has a substantial capacity for non-photochemical quenching (NPQ) of chlorophyll fluorescence. In contrast, D. antarctica has up to 30% of its photosynthetic electron transport being linked to oxygen, but has a substantially lower capacity for NPQ. Thus, these plants rely on contrasting photoprotective mechanisms to cope with the Antarctic environment. Both plants seem to use cyclic electron flow associated with PSI, however, this is activated at a lower irradiance in C. quitensis than in D. antarctica.     

Carbon sink limitation and frost tolerance control performance of the tree <Emphasis Type="Italic">Kageneckia angustifolia</Emphasis> D. Don (Rosaceae) at the treeline in central Chile

The decrease in temperature with increasing elevation may determine the altitudinal tree distribution in different ways: affecting survival through freezing temperatures, by a negative carbon balance produced by lower photosynthetic rates, or by limiting growth activity. Here we assessed the relative importance of these direct and indirect effects of altitudinal decrease in temperature in determining the treeline in central Chile (33°S) dominated by Kageneckia angustifolia. We selected two altitudes (2000 and 2200 m a.s.l.) along the treeline ecotone. At each elevation, leaf non-structural carbohydrates (NSC) and gas exchange parameters were measured on five individuals during the growing season. We also determined the cold resistance of K.␣angustifolia, by measuring temperatures that cause 50% seedling mortality (LT₅₀) and ice nucleation (IN). No differences in net photosynthesis were found between altitudes. Although no differences were detected on NSC concentration on a dry matter basis between 2000 and 2200 m, when NSC concentration was expressed on a leaf area basis, higher contents were found at the higher elevation. Thus, carbon sink limitations may occur at the K. angustifolia’s upper altitudinal limit. For seedlings derived from seeds collected at the 2200 m, LT₅₀ of cold-acclimated and non-acclimated plants were −9.5 and −7 °C, respectively. However, temperatures as low as −10 °C can frequently occur at this altitude during the end of winter. Therefore, low temperature injury of seedlings seems also be involved in the treeline formation in this species. Hence, a confluence of global (carbon sink limitation) and regional (freezing tolerance) mechanisms explains the treeline formation in the Mediterranean-type climate zone of central Chile.     

Inhibition of ATPase from chloroplasts by a hydroxamic acid from the gramineae

-DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), a hydroxamic acid from the Gramineae involved in the resistance of cereals to aphids, inhibits     

Leaf functional and micro-morphological photoprotective attributes in two ecotypes of Colobanthus quitensis from the Andes and Maritime Antarctic

Colobanthus quitensis (Kunth) Bartl. (Cariophyllaceae) is distributed from Mexico to the Maritime Antarctic. It grows forming inconspicuous populations in humid and cold sites along high elevations in the Andes Mountains. Mediterranean Andes is characterized by a wider oscillation of diurnal and seasonal temperature, while the Maritime Antarctic is characterized by permanent low temperatures. Both places may experience high irradiance during sunny days (reaching up to 2,000 μmol photons m⁻² s⁻¹); however, the frequency of sunny days in the Maritime Antarctica is significantly lower (less than 20% of the whole growing season). We study whether acclimation to each environment relies on different photoprotective mechanisms. The Andean ecotype that has a longer growing season and a higher light integral reduces light absorption by the development of smaller chloroplasts with lower stacking granum area and down-regulation of Lhcb2. It also enhances the dissipation of the excess of absorbed energy by higher level of de-epoxidation of xanthophylls pool. On the other hand, the Antarctic ecotype which has developed under a shorter growing season, with lower total irradiance and continuous low temperatures, maximizes photochemical process even at low temperatures and it has a lower light-harvesting/core complex ratio and higher level of photoprotection supplied by an unusually high β-carotene and xanthophylls cycle pool. It resembles a well full light acclimated plant, probably due to higher excitation pressure imposed by lower temperature even at moderate irradiance. It is suggested that the biochemical plasticity of this species, highlighted by the development of these different strategies, is essential to cope successfully with these particular environments.     

Effects of a severe drought on Quercus ilex radial growth and xylem anatomy

We assessed the response of Quercus ilex subsp. ballota to the severe summer drought recorded in 1994 in NE Spain through the study of changes in radial growth and wood anatomy. We selected a coppice stand in the Iberian Peninsula, which is characterized by a Mediterranean climate under continental influence. We measured internode length, tree-ring width, mean and maximum vessel diameter, and vessel density for 1981–1997. The annual predicted hydraulic conductance (K_h) was calculated following Hagen-Poisseuille's law. We compared the tree-ring width, vessel diameter and K_h of Q. ilex subsp. ballota and co-existing ring-porous oaks (Q. faginea, Q. pyrenaica) for a dry summer (1994) and a wet summer (1997). To evaluate the drought-resistance of xylem for Q. ilex subsp. ballota (dominant under continental conditions) and Q. ilex subsp. ilex (dominant in mild areas) we determined vulnerability curves. Dimensionless indices of internode length, tree-ring width, and vessel density were compared with climatic data (monthly total precipitation and mean temperature) using correlation analyses. Internode length, tree-ring width, K_h, and mean and maximum vessel diameter declined in 1994. According to vulnerability curves, Q. ilex subsp. ballota showed a greater drought resistance than Q. ilex subsp. ilex. During the year of growth, we found a positive influence of January and June–August precipitation on the internode length, tree-ring width, and vessel density. The response of Q. ilex subsp. ballota radial-growth to summer drought was comparable to that of Q. faginea latewood. Overall, growth and wood anatomy of Q. ilex subsp. ballota showed a plastic response to drought.