Cold-induced changes affect survival after exposure to vitrification solution during cryopreservation in the south-west Australian Mediterranean climate species Lomandra sonderi (Asparagaceae)

There is limited knowledge of the effects of exposure to low temperatures in the unique Mediterranean climate plant species of Western Australia. We have thus investigated the effect of low temperature on cryogenic tolerance in Lomandra sonderi, an endemic perennial species of southwest Western Australia. Lomandra sonderi plants were preconditioned in tissue culture at constant 23 °C (12 h light/dark cycle) or alternating 20/?1 °C (16 h light and 8 h dark cycle). Shoot tips from both conditions were analysed for their phospholipid, sterol and soluble sugar compositions. Shoot tips were also cryoexposed via a droplet-vitrification protocol. Survival in both preconditioning regimes for cryoexposed and non-cryoexposed samples was the same, but plants from the 20/?1 °C regime displayed an improved tolerance to the overall cryopreservation process in both cryoexposed and non-cryoexposed samples, thereby eliminating exposure to liquid nitrogen as a primary cause of reduced post-cryogenic viability. Preconditioning of in vitro shoots of L. sonderi at 20/?1 °C induced significant increases in phosphatidylcholine (from 7.30 ± 3.46 to 22.2 ± 7.80 ng mg^?1 FW) and increases in several soluble sugars (fructose, galactose, glucose, sucrose) compared to shoots incubated at 23 °C—changes consistent with known cold acclimation responses in plant species generally—but sterol content remained largely unchanged. Analysis of electrolyte leakage in shoot tips from both preconditioning regimes generated a significantly lower LT₅₀ value in the 20/?1 °C samples (?5.45 ± 0.53 °C) over the 23 °C samples (?2.5 ± 0.08 °C). Increased tolerance to cryoexposure in L. sonderi appears to lie mainly with acclimation-induced changes in membrane composition and promotion of membrane stability and hence increased resistance to freeze damage.     

Heat tolerance and cold tolerance of cultivated potatoes measured by the chlorophyll-fluorescence method

Heat and cold tolerances were determined for 13 clones of the commonly cultivated potato, Solanum tuberosum L. Five clones were considered to be adapted to warm climates and the others to cool climates only in terms of their ability to produce tubers. The decrease in the rate of the induced rise in chlorophyll fluorescence after heating leaves at 41°C for 10 min was used to measure relative heat tolerance, and the decrease following chilling at 0°C was used to measure relative cold tolerance. The warm-adapted clones all showed enhanced heat tolerance compared with the cool-adapted clones. Higher heat tolerance was also correlated with a greater tolerance towards a cold stress of 0°C and it is suggested that the warm-adapted clones were selections showing an increased generalized capacity to withstand environmental stresses of several kinds rather than a specific genotypic adaptation to tolerate warm temperatures. Heat and cold tolerances were also determined for several other species of potato cultivated in the Andean region of South America. Of these, S. phureja, which is found at low altitudes on the eastern slopes of the Andes, showed a tolerance to heat comparable to that of the warm-adapted clones of the common potato, the two most heat tolerant of which contained some phureja in their parentage. Diploid and triploid species of cultivated potatoes were considerably more cold tolerant than the clones of the common potato, a tetraploid. The genetic variability for heat and cold tolerance in cultivated and wild potatoes is discussed in relation to increasing the tolerance of the potato to these stresses.     

Expression of hsp70, hsp100 and ubiquitin in Aloe barbadensis Miller under direct heat stress and under temperature acclimation conditions

Key message

The study determined the tolerance of Aloe vera to high temperature, focusing on the expression of hsp70 , hsp100 and ubiquitin genes. These were highly expressed in plants acclimated at 35 °C prior to a heat shock of 45 °C.

Abstract

Aloe barbadensis Miller (Aloe vera), a CAM plant, was introduced into Chile in the semiarid IV and III Regions, which has summer diurnal temperature fluctuations of 25 to 40 °C and annual precipitation of 40 mm (dry years) to 170 mm (rainy years). The aim of this study was to investigate how Aloe vera responds to water and heat stress, focusing on the expression of heat shock genes (hsp70, hsp100) and ubiquitin, which not studied before in Aloe vera. The LT₅₀ of Aloe vera was determined as 53.2 °C. To study gene expression by semi-quantitative RT-PCR, primers were designed against conserved regions of these genes. Sequencing the cDNA fragments for hsp70 and ubiquitin showed a high identity, over 95 %, with the genes from cereals. The protein sequence of hsp70 deduced from the sequence of the cDNA encloses partial domains for binding ATP and the substrate. The protein sequence of ubiquitin deduced from the cDNA encloses a domain for interaction with the enzymes E2, UCH and CUE. The expression increased with temperature and water deficit. Hsp70 expression at 40–45 °C increased 50 % over the controls, while the expression increased by 150 % over the controls under a water deficit of 50 % FC. The expression of all three genes was also studied under 2 h of acclimation at 35 or 40 °C prior to a heat shock at 45 °C. Under these conditions, the plants showed greater expression of all genes than when they were subjected to direct heat stress.     

Human recombinant factor VIIa may improve heat intolerance in mice by attenuating hypothalamic neuronal apoptosis and damage

Intolerance to heat exposure is believed to be associated with hypothalamo-pituitary-adrenocortical (HPA) axis impairment [reflected by decreases in blood concentrations of both adrenocorticotrophic-hormone (ACTH) and corticosterone]. The purpose of this study was to determine the effect of human recombinant factor VIIa (rfVIIa) on heat intolerance, HPA axis impairment, and hypothalamic inflammation, ischemic and oxidative damage, and apoptosis in mice under heat stress. Immediately after heat stress (41.2 °C for 1 h), mice were treated with vehicle (1 mL/kg of body weight) or rfVIIa (65–270 µg/kg of body weight) and then returned to room temperature (26 °C). Mice still alive on day 4 of heat exposure were considered survivors. Cellular ischemia markers (e.g., glutamate, lactate-to-pyruvate ratio), oxidative damage markers (e.g., nitric oxide metabolite, hydroxyl radials), and pro-inflammatory cytokines (e.g., interleukin-6, interleukin-1β, tumor necrosis factor-α) in hypothalamus were determined. In addition, blood concentrations of both ACTH and corticosterone were measured. Hypothalamic cell damage was assessed by determing the neuronal damage scores, whereas the hypothalamic cell apoptosis was determined by assessing the numbers of cells stained with terminal deoxynucleotidyl transferase-mediated αUTP nick-end labeling, caspase-3-positive cells, and platelet endothelial cell adhesion molecula-1-positive cells in hypothalamus. Compared with vehicle-treated heated mice, rfVIIa-treated heated mice had significantly higher fractional survival (8/10 vs 1/10), lesser thermoregulatory deficit (34.1 vs 24.8 °C), lesser extents of ischemic, oxidative, and inflammatory markers in hypothalamus, lesser neuronal damage scores and apoptosis in hypothalamus, and lesser HPA axis impairment. Human recombinant factor VIIa appears to exert a protective effect against heatstroke by attenuating hypothalamic cell apoptosis (due to ischemic, inflammatory, and oxidative damage) in mice.