Drosophila melanogaster is a chill-susceptible insect. Previous studies on this fly focused on acute direct chilling injury during cold shock and showed that lower lethal temperature (LLT, approximately −5°C) exhibits relatively low plasticity and that acclimations, both rapid cold hardening (RCH) and long-term cold acclimation, shift the LLT by only a few degrees at the maximum. We found that long-term cold acclimation considerably improved cold tolerance in fully grown third-instar larvae of D. melanogaster. A comparison of the larvae acclimated at constant 25°C with those acclimated at constant 15°C followed by constant 6°C for 2 d (15°C→6°C) showed that long-term cold acclimation extended the lethal time for 50% of the population (Lt₅₀) during exposure to constant 0°C as much as 630-fold (from 0.137 h to 86.658 h). Such marked physiological plasticity in Lt₅₀ (in contrast to LLT) suggested that chronic indirect chilling injury at 0°C differs from that caused by cold shock. Long-term cold acclimation modified the metabolomic profiles of the larvae. Accumulations of proline (up to 17.7 mM) and trehalose (up to 36.5 mM) were the two most prominent responses. In addition, restructuring of the glycerophospholipid composition of biological membranes was observed. The relative proportion of glycerophosphoethanolamines (especially those with linoleic acid at the sn-2 position) increased at the expense of glycerophosphocholines. Third-instar larvae of D. melanogaster improved their cold tolerance in response to long-term cold acclimation and showed metabolic potential for the accumulation of proline and trehalose and for membrane restructuring.

Among vertebrates, only a few species of amphibians and reptiles tolerate the formation of ice crystals in their body fluids. Freeze tolerance is much more widespread in invertebrates, especially in overwintering insects. Evolutionary adaptations for freeze tolerance are considered to be highly complex. Here we show that surprisingly simple laboratory manipulations can change the chill susceptible insect to the freeze tolerant one. Larvae of Drosophila melanogaster , a fruit fly of tropical origin with a weak innate capacity to tolerate mild chilling, can survive when approximately 50% of their body water freezes. To achieve this goal, synergy of two fundamental prerequisites is required: ( i ) shutdown of larval development by exposing larvae to low temperatures (dormancy) and ( ii ) incorporating the free amino acid proline in tissues by feeding larvae a proline-augmented diet (cryopreservation).

The larva of the drosophilid fly, Chymomyza costata , is probably the most complex metazoan organism that can survive submergence in liquid nitrogen (-196 °C) in a fully hydrated state. We examined the associations between the physiological and biochemical parameters of differently acclimated larvae and their freeze tolerance. Entering diapause is an essential and sufficient prerequisite for attaining high levels of survival in liquid nitrogen (23% survival to adult stage), although cold acclimation further improves this capacity (62% survival). Profiling of 61 different metabolites identified proline as a prominent compound whose concentration increased from 20 to 147 mM during diapause transition and subsequent cold acclimation. This study provides direct evidence for the essential role of proline in high freeze tolerance. We increased the levels of proline in the larval tissues by feeding larvae proline-augmented diets and found that this simple treatment dramatically improved their freeze tolerance. Cell and tissue survival following exposure to liquid nitrogen was evident in proline-fed nondiapause larvae, and survival to adult stage increased from 0% to 36% in proline-fed diapause-destined larvae. A significant statistical correlation was found between the whole-body concentration of proline, either natural or artificial, and survival to the adult stage in liquid nitrogen for diapause larvae. Differential scanning calorimetry analysis suggested that high proline levels, in combination with a relatively low content of osmotically active water and freeze dehydration, increased the propensity of the remaining unfrozen water to undergo a glass-like transition (vitrification) and thus facilitated the prevention of cryoinjury.

Summary: Target metabolic and large‐scale transcriptomic analyses of tobacco (Nicotiana tabacum L.) Bright Yellow‐2 (BY‐2) cells were employed to identify novel gene(s) involved in methyl jasmonate (MJ)‐dependent function in plants. At the metabolic level, we describe the specific accumulation of several phenylpropanoid–polyamine conjugates in MJ‐treated BY‐2 cells. Furthermore, global gene expression analysis of MJ‐treated cells using a 16K cDNA microarray containing expressed sequence tags (ESTs) from BY‐2 cells revealed 828 genes that were upregulated by MJ treatment within 48 h. Using time‐course expression data we identified a novel MJ‐inducible R2R3 MYB‐type transcription factor (NtMYBJS1) that was co‐expressed in a close temporal pattern with the core phenylpropanoid genes phenylalanine ammonia‐lyase (PAL) and 4‐coumarate:CoA ligase (4CL). Overexpression of NtMYBJS1 in tobacco BY‐2 cells caused accumulation of specific phenylpropanoid conjugates in the cells. Subsequent microarray analysis of NtMYBJS1 transgenic lines revealed that a limited number of genes, including PAL and 4CL, were specifically induced in the presence of the NtMYBJS1 transgene. These results, together with results of both antisense expression analysis and of gel mobility shift assays, strongly indicate that the NtMYBJS1 protein functions in tobacco MJ signal transduction, inducing phenylpropanoid biosynthetic genes and the accumulation of phenylpropanoid–polyamine conjugates during stress.