How consistent are the transcriptome changes associated with cold acclimation in two species of the Drosophila virilis group?

This work was financially support by a Marie Curie Initial Training Network grant, “Understanding the evolutionary origin of biological diversity” (ITN-2008–213780 SPECIATION), grants from the Academy of Finland to A.H. (project 132619) and M.K. (projects 268214 and 272927), a grant from NERC, UK to M.G.R. (grant NE/J020818/1), and NERC, UK PhD studentship to D.J.P. (NE/I528634/1).For many organisms the ability to cold acclimate with the onset of seasonal cold has major implications for their fitness. In insects, where this ability is widespread, the physiological changes associated with increased cold tolerance have been well studied. Despite this, little work has been done to trace changes in gene expression during cold acclimation that lead to an increase in cold tolerance. We used an RNA-Seq approach to investigate this in two species of the Drosophila virilis group. We found that the majority of genes that are differentially expressed during cold acclimation differ between the two species. Despite this, the biological processes associated with the differentially expressed genes were broadly similar in the two species. These included: metabolism, cell membrane composition, and circadian rhythms, which are largely consistent with previous work on cold acclimation/cold tolerance. In addition, we also found evidence of the involvement of the rhodopsin pathway in cold acclimation, a pathway that has been recently linked to thermotaxis. Interestingly, we found no evidence of differential expression of stress genes implying that long-term cold acclimation and short-term stress response may have a different physiological basis.PostprintPeer reviewe

Identification and expression analysis of CBF/DREB1 and COR15 genes in mutants of Brassica oleracea var. botrytis with enhanced proline production and frost resistance.

Frost resistant mutants of Brassica oleracea var. botrytis were investigated for the presence of CBF/DREB1 and COR15a gene products and induced frost resistance. Total RNA of clones was isolated after 3 h, 6 h, 24 h and 14 d acclimation at 4 °C and proteins and free proline were isolated after 14 d acclimation. cDNA was produced using RT-PCR and the first CBF gene in B. oleracea detected and did quantify. Through SDS-PAGE and Western blotting, the COR15a protein was detected for the first time in B. oleracea. The results confirmed the first report of the presence of BoCBF/DREB1 in B. oleracea and this only appeared under cold acclimation. The sequence analysis of predicted amino acids revealed a very high homology (90%) with CBF sequences of other Brassica species (BnCBF5/DREB1, BrDREB1 and BjDREB1B) and homology reduced to 67% when compared to plants other than Brassicas. BoCBF/DREB1 transcript levels increased up to 24 h acclimation and then declined. Some mutants showed BoCBF/DREB1 expression at 3 h while others only after 6 h and 24 h acclimation. The genotypes showed positive significant correlation between BoCBF/DREB1 expression and frost resistance (R(2) = 0.9343). The proline level under acclimation increased about 8 fold and demonstrated positive and significant correlation with BoCBF/DREB1 expression. Proline also showed positive and significant correlation with frost resistance under cold acclimation but very not under non-acclimation. All clones were positive for COR15a protein after 14 d cold acclimation and expression correlated with frost resistance. Under non-acclimation COR15a was constitutively expressed in 3 mutants

Plasma membrane lipid remodeling during cold acclimation is mediated by the ER-PM contact sites-localized synaptotagmins 1 and 3

Cold acclimation is the capacity of certain plants to increase their freezing tolerance in response to a period of low non-freezing temperatures. Cold acclimation involves a series of biochemical and physiological adaptations, including a deep transcriptional reprogramming and drastic changes in the lipid composition of cellular membranes in order to prevent the freeze-induced damage (1). While a profound knowledge has been acquired on the regulation of gene expression triggered by cold-acclimation, very little is known about the mechanisms governing the cold-induced changes in membranes’ lipid composition. In this study we report that in Arabidopsis, the constitutively expressed Synaptotagmin 1 (SYT1) and the cold-induced homolog Synaptotagmin 3 (SYT3) are essential for cold- acclimated freezing tolerance and for the lipid remodelling of the plasma membrane during cold-acclimation. SYT1 and SYT3 are phospholipid-binding proteins located in Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PMcs), conserved structures defined as regions of the cortical ER in close apposition to the PM (2). ER-PMcs facilitate the non-vesicular lipid transport between ER and PM in yeast and mammals, and are essential for lipid homeostasis (3). In contrast to the high and ubiquitous SYT1 expression, SYT3 expression is low and mainly restricted to meristemoids, young stomata, and old primary root. TIRF microscopy analyses show that during cold acclimation there is an increase of SYT1::SYT1:GFP and SYT3::SYT3:GFP signals as spots at the PM. High-resolution lipidome analyses show the over-accumulation of phosphatidylinositols phosphate (PIPs) and glycerolipids in vivo in syt1 and specially syt1/syt3 mutant plants compared to WT in one-week cold-acclimated plants. Interestingly, protein-lipid overlay assays (membrane-strips and PIP-strips) reveal PIPs and glycerolipids as major interactors for both, SYT1 and SYT3. Here we show that 1) Arabidopsis SYT1 and SYT3 are induced by cold, 2) SYT1 and SYT3 localize to ER-PMcs, 3) the specific lipids that directly interact with SYT1 and SYT3 accumulate in syt1/syt3 mutant after cold acclimation, and 4) syt1/syt3 show reduced cold acclimated freezing tolerance. We propose that SYT1 and SYT3 have essential roles in ER-PMcs mediated lipid remodelling during cold acclimation, which in turn leads to freezing tolerance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Water stress is a component of cold acclimation process essential for inducing full freezing tolerance in strawberry

The factors involved in cold acclimation process and their role in inducing freezing tolerance were studied in strawberry (Fragaria X ananassa) plants. The results show that low temperature and water stress are two key components of cold acclimation, in that low temperature typically induced water stress in the plants. After a 2-week exposure of plants to 3/1°C (day/night temperature), the leaf water potential decreased markedly to below -1.6 MPa. While both of these components contribute significantly to the induction of freezing tolerance, water stress is a dominant factor in inducing freezing tolerance, contributing roughly to 56% of freezing tolerance acquired by natural cold acclimation. Typical cold acclimation treatment of plants for 2 weeks increased their freezing tolerance by about 14°C to -20.7°C while the same treatment, in the absence of the accompanying water stress, increased their freezing tolerance only by 5°C, which indicates the importance of water stress during cold acclimation. Furthermore, both low temperature and water stress independently induced the orthologs of cold-responsive genes, COR47 and COR78, however, stronger expression of these genes was observed in response to cold acclimating conditions. Thus, these results show that both of these factors are essential elements of cold acclimation process and play an important role in inducing freezing tolerance in strawberry plants

The effect of cold priming on the fitness of Arabidopsis thaliana accessions under natural and controlled conditions

Priming improves an organism's performance upon a future stress. To test whether cold priming supports protection in spring and how it is affected by cold acclimation, we compared seven Arabidopsis accessions with different cold acclimation potentials in the field and in the greenhouse for growth, photosynthetic performance and reproductive fitness in March and May after a 14 day long cold-pretreatment at 4 °C. In the plants transferred to the field in May, the effect of the cold pretreatment on the seed yield correlated with the cold acclimation potential of the accessions. In the March transferred plants, the reproductive fitness was most supported by the cold pretreatment in the accessions with the weakest cold acclimation potential. The fitness effect was linked to long-term effects of the cold pretreatment on photosystem II activity stabilization and leaf blade expansion. The study demonstrated that cold priming stronger impacts on plant fitness than cold acclimation in spring in accessions with intermediate and low cold acclimation potential

Inducing cold-sensitivity in the frigophilic fly Drosophila montana by RNAi

The work was supported by CNPq (Fellowship to FMV) and a NERC Studentship to DJP.Cold acclimation is a critical physiological adaptation for coping with seasonal cold. By increasing their cold tolerance individuals can remain active for longer at the onset of winter and can recover more quickly from a cold shock. In insects, despite many physiological studies, little is known about the genetic basis of cold acclimation. Recently, transcriptomic analyses in Drosophila virilis and D.montana revealed candidate genes for cold acclimation by identifying genes upregulated during exposure to cold. Here, we test the role of myo-inositol-1-phosphate synthase (Inos), in cold tolerance in D. montana using an RNAi approach. D. montana has a circumpolar distribution and overwinters as an adult in northern latitudes with extreme cold. We assessed cold tolerance of dsRNA knock-down flies using two metrics: chill-coma recovery time (CCRT) and mortality rate after cold acclimation. Injection of dsRNAInos did not alter CCRT,either overall or in interaction with the cold treatment, however it did induced cold specific mortality, with high levels of mortality observed in injected flies acclimated at 5°C but not at 19°C. Overall, injection with dsRNAInos induced a temperature sensitive mortality rate of over 60% in this normally cold-tolerant species. qPCRanalysis confirmed that dsRNA injection successfully reduced gene expression of Inos. Thus, our results demonstrate the involvement of Inos in increasing cold tolerance in D. montana. The potential mechanisms involved by which Inos increases cold tolerance are also discussed.Publisher PDFPeer reviewe

Effects of Temperature on Organ Masses and Digestive Tract Morphology in Apodemus chevrieri from Hengduan Mountain Region

In order to investigate the changes of plasticity of visceral organs and digestive tract morphology in Apodemus chevrieri which inhabit in Hengduan mountain region, the organ masses and digestive tract which include the changes of heart, lung, liver , spleen and kidney as well as the length and weight of stomach, small intestine, large intestine and cecum were measured during cold and warm acclimation. The results showed that the weight of heart and liver of cold acclimation group were obviously higher than that of warm acclimation group. The weight and length of small intestine showed significant differences between two groups. All of the results indicated that A. chevrieri maintain their normal life activities by increasing the weight of related organ masses and adjusting the weight and length of small intestine under cold temperature

Interaction of Temperature and Light in the Development of Freezing Tolerance in Plants

Abstract Freezing tolerance is the result of a wide range of physical and biochemical processes, such as the induction of antifreeze proteins, changes in membrane composition, the accumulation of osmoprotectants, and changes in the redox status, which allow plants to function at low temperatures. Even in frost-tolerant species, a certain period of growth at low but nonfreezing temperatures, known as frost or cold hardening, is required for the development of a high level of frost hardiness. It has long been known that frost hardening at low temperature under low light intensity is much less effective than under normal light conditions; it has also been shown that elevated light intensity at normal temperatures may partly replace the cold-hardening period. Earlier results indicated that cold acclimation reflects a response to a chloroplastic redox signal while the effects of excitation pressure extend beyond photosynthetic acclimation, influencing plant morphology and the expression of certain nuclear genes involved in cold acclimation. Recent results have shown that not only are parameters closely linked to the photosynthetic electron transport processes affected by light during hardening at low temperature, but light may also have an influence on the expression level of several other cold-related genes; several cold-acclimation processes can function efficiently only in the presence of light. The present review provides an overview of mechanisms that may explain how light improves the freezing tolerance of plants during the cold-hardening period

Intraspecific variation in thermal acclimation and tolerance between populations of the winter ant, Prenolepis imparis.

Thermal phenotypic plasticity, otherwise known as acclimation, plays an essential role in how organisms respond to short-term temperature changes. Plasticity buffers the impact of harmful temperature changes; therefore, understanding variation in plasticity in natural populations is crucial for understanding how species will respond to the changing climate. However, very few studies have examined patterns of phenotypic plasticity among populations, especially among ant populations. Considering that this intraspecies variation can provide insight into adaptive variation in populations, the goal of this study was to quantify the short-term acclimation ability and thermal tolerance of several populations of the winter ant, Prenolepis imparis. We tested for correlations between thermal plasticity and thermal tolerance, elevation, and body size. We characterized the thermal environment both above and below ground for several populations distributed across different elevations within California, USA. In addition, we measured the short-term acclimation ability and thermal tolerance of those populations. To measure thermal tolerance, we used chill-coma recovery time (CCRT) and knockdown time as indicators of cold and heat tolerance, respectively. Short-term phenotypic plasticity was assessed by calculating acclimation capacity using CCRT and knockdown time after exposure to both high and low temperatures. We found that several populations displayed different chill-coma recovery times and a few displayed different heat knockdown times, and that the acclimation capacities of cold and heat tolerance differed among most populations. The high-elevation populations displayed increased tolerance to the cold (faster CCRT) and greater plasticity. For high-temperature tolerance, we found heat tolerance was not associated with altitude; instead, greater tolerance to the heat was correlated with increased plasticity at higher temperatures. These current findings provide insight into thermal adaptation and factors that contribute to phenotypic diversity by revealing physiological variance among populations

In the quest for a cold tolerant variety - gene expression profile analysis of cold stressed oat and rice

Cold acclimation is a process which increases the freezing tolerance of an organism, after exposure to low, non-freezing temperatures. The acclimation ensures that cold tolerant species can endure harsh winter conditions, by preparing them to sub-zero temperatures. Cold-sensitive plants such as oat and rice have limited abilities to cold acclimate and are therefore easily damaged during winter time. The development of more tolerant varieties by using biotechnological methods is desirable, since the yields are expected to improve due to a prolonged vegetation period. However, in order to apply such methods, more knowledge about the underlying mechanisms regulating the cold tolerance and acclimation is required. One step in this direction is to analyze gene expression data generated from cold stressed oat (Part I) and rice plants (Part II). The focus of this thesis is, consequently, analysis of expression profiling data, which was generated using the EST sequencing and cDNA microarray technologies. The results show that both oat and rice are cold responsive, with many of the previously identified cold regulated genes having a counterpart in these species. In rice, however, the response is less dynamic than in the model organism Arabidopsis thaliana and this may explain its inability to fully cold acclimate. Additionally, the work in this thesis focuses on evaluating if small-scale EST sets can be used for ‘digital-Northern’, in order to identify genes that are involved in the regulation of the cold stress response. The results show that small-scaled EST sets are not optimal for such an analysis, since the method detected only a portion of cold responsive genes represented in the sets. This has to due with the inherent properties of EST data and limitations in the analysis steps of the sequences. The work also concerns the identification of cis-elements coupled to transcription factors prominent in the regulation of the response. Since cold acclimation is a quantitative trait the response and regulation of cold stress is under combinatorial control of several transcription factors and the results show that this should be taken into account when identifying binding sites