Distinct cold tolerance traits independently vary across genotypes in Drosophila melanogaster

Cold tolerance, the ability to cope with low temperature stress, is a critical adaptation in thermally variable environments. An individual's cold tolerance comprises several traits including minimum temperatures for growth and activity, ability to survive severe cold, and ability to resume normal function after cold subsides. Across species, these traits are correlated, suggesting they were shaped by shared evolutionary processes or possibly share physiological mechanisms. However, the extent to which cold tolerance traits and their associated mechanisms covary within populations has not been assessed. We measured five cold tolerance traits—critical thermal minimum, chill coma recovery, short- and long-term cold tolerance, and cold-induced changes in locomotor behavior—along with cold-induced expression of two genes with possible roles in cold tolerance (heat shock protein 70 and frost)—across 12 lines of Drosophila melanogaster derived from a single population. We observed significant genetic variation in all traits, but few were correlated across genotypes, and these correlations were sex-specific. Further, cold-induced gene expression varied by genotype, but there was no evidence supporting our hypothesis that cold-hardy lines would have either higher baseline expression or induction of stress genes. These results suggest cold tolerance traits possess unique mechanisms and have the capacity to evolve independently.     

Evolutionary engineering reveals divergent paths when yeast is adapted to different acidic environments

Tolerance of yeast to acid stress is important for many industrial processes including organic acid production. Therefore, elucidating the molecular basis of long term adaptation to acidic environments will be beneficial for engineering production strains to thrive under such harsh conditions. Previous studies using gene expression analysis have suggested that both organic and inorganic acids display similar responses during short term exposure to acidic conditions. However, biological mechanisms that will lead to long term adaptation of yeast to acidic conditions remains unknown and whether these mechanisms will be similar for tolerance to both organic and inorganic acids is yet to be explored. We therefore evolved Saccharomyces cerevisiae to acquire tolerance to HCl (inorganic acid) and to 0.3 M L-lactic acid (organic acid) at pH 2.8 and then isolated several low pH tolerant strains. Whole genome sequencing and RNA-seq analysis of the evolved strains revealed different sets of genome alterations suggesting a divergence in adaptation to these two acids. An altered sterol composition and impaired iron uptake contributed to HCl tolerance whereas the formation of a multicellular morphology and rapid lactate degradation was crucial for tolerance to high concentrations of lactic acid. Our findings highlight the contribution of both the selection pressure and nature of the acid as a driver for directing the evolutionary path towards tolerance to low pH. The choice of carbon source was also an important factor in the evolutionary process since cells evolved on two different carbon sources (raffinose and glucose) generated a different set of mutations in response to the presence of lactic acid. Therefore, different strategies are required for a rational design of low pH tolerant strains depending on the acid of interest.     

植物逆境胁迫耐受性功能基因组研究进展

为了更加高效地利用基因工程技术提高植物对逆境胁迫的耐受性,需要在全基因组水平上对植物逆境胁迫耐受性的复杂机制进行整合性研究.植物逆境胁迫耐受性功能基因组的研究可概括为：利用胁迫特异性的表达序列标签(EST)及cDNA微阵列（或基因芯片）技术筛选与胁迫相关的候选基因,然后利用反向遗传学等技术对候选基因的功能进行研究,利用酵母双杂交、正向遗传学等技术对基因及基因产物间的相互关系进行研究.通过这些研究可以全面地了解植物对胁迫（渗透、干旱、极端温度）响应的复杂机制和相互作用以及相应的信号转导途径,从而为更加高效地利用基因工程技术提高植物对逆境胁迫的耐受性奠定基础.     

Functional gene-mining for salt-tolerance genes with the power of Arabidopsis

Here we report on a functional gene-mining method developed to isolate stress tolerance genes without any prior knowledge of the genome or genetic mapping of the source germplasms. The feasibility of this approach was demonstrated by isolating novel salt stress tolerance genes from salt cress (Thellungiella halophila), an extremophile that is adapted to a harsh saline environment and a close relative of the model plant Arabidopsis thaliana. This gene-mining method is based on the expression of salt cress cDNA libraries in Arabidopsis. A cDNA expression library of the source germplasm, salt cress, was constructed and used to transform Arabidopsis via Agrobacterium-mediated gene transfer. A transgenic seed library consisting of >125,000 independent lines was generated and screened for salt-tolerant lines via a high-throughput genetic screen. A number of salt-tolerant lines were isolated, and the salt cress cDNAs were identified by PCR amplification and sequencing. Among the genes isolated, several novel small protein-encoding genes were discovered. The homologs of these genes in Arabidopsis have not been experimentally analyzed, and their functions remain unknown. The function of two genes isolated by this method, ST6-66 and ST225, and their Arabidopsis homologs, were investigated in Arabidopsis using gain- and loss-of-function analyses, and their importance in salt tolerance was demonstrated. Thus, our functional gene-mining method was validated by these results. Our method should be applicable for the functional mining of stress tolerance genes from various germplasms. Future improvements of the method are also discussed.     

Evolution of Robustness to Protein Mistranslation by Accelerated Protein Turnover

Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome–mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that translational fidelity and the ubiquitin-proteasome system are functionally linked to each other and may, therefore, co-evolve in nature.     

Chromosome-level genome assembly of a xerophytic plant,Haloxylon ammodendron

Haloxylon ammodendron is a xerophytic perennial shrub or small tree that has a high ecological value in anti-desertification due to its high tolerance to drought and salt stress. Here, we report a high-quality, chromosome-level genome assembly of H. ammodendron by integrating PacBio’s high-fidelity sequencing and Hi-C technology. The assembled genome size was 685.4 Mb, of which 99.6% was assigned to nine pseudochromosomes with a contig N50 value of 23.6 Mb. Evolutionary analysis showed that both the recent substantial amplification of long terminal repeat retrotransposons and tandem gene duplication may have contributed to its genome size expansion and arid adaptation. An ample amount of low-GC genes was closely related to functions that may contribute to the desert adaptation of H. ammodendron. Gene family clustering together with gene expression analysis identified differentially expressed genes that may play important roles in the direct response of H. ammodendron to water-deficit stress. We also identified several genes possibly related to the degraded scaly leaves and well-developed root system of H. ammodendron. The reference-level genome assembly presented here will provide a valuable genomic resource for studying the genome evolution of xerophytic plants, as well as for further genetic breeding studies of H. ammodendron.     

Joint genetic and network analyses identify loci associated with root growth under NaCl stress in Arabidopsis thaliana

Plants have evolved a series of tolerance mechanisms to saline stress, which perturbs physiological processes throughout the plant. To identify genetic mechanisms associated with salinity tolerance, we performed linkage analysis and genome‐wide association study (GWAS) on maintenance of root growth of Arabidopsis thaliana in hydroponic culture with weak and severe NaCl toxicity. The top 200 single‐nucleotide polymorphisms (SNPs) determined by GWAS could cumulatively explain approximately 70% of the variation observed at each stress level. The most significant SNPs were linked to the genes of ATP‐binding cassette B10 and vacuolar proton ATPase A2. Several known salinity tolerance genes such as potassium channel KAT1 and calcium sensor SOS3 were also linked to SNPs in the top 200. In parallel, we constructed a gene co‐expression network to independently verify that particular groups of genes work together to a common purpose. We identify molecular mechanisms to confer salt tolerance from both predictable and novel physiological sources and validate the utility of combined genetic and network analysis. Additionally, our study indicates that the genetic architecture of salt tolerance is responsive to the severity of stress. These gene datasets are a significant information resource for a following exploration of gene function.     

Ploidy and recombination proficiency shape the evolutionary adaptation to constitutive DNA replication stress

In haploid budding yeast, evolutionary adaptation to constitutive DNA replication stress alters three genome maintenance modules: DNA replication, the DNA damage checkpoint, and sister chromatid cohesion. We asked how these trajectories depend on genomic features by comparing the adaptation in three strains: haploids, diploids, and recombination deficient haploids. In all three, adaptation happens within 1000 generations at rates that are correlated with the initial fitness defect of the ancestors. Mutations in individual genes are selected at different frequencies in populations with different genomic features, but the benefits these mutations confer are similar in the three strains, and combinations of these mutations reproduce the fitness gains of evolved populations. Despite the differences in the selected mutations, adaptation targets the same three functional modules in strains with different genomic features, revealing a common evolutionary response to constitutive DNA replication stress.     

Recent genome duplications facilitate the phenotypic diversity of Hb repertoire in the Cyprinidae

Whole-genome duplications(WGDs) are an important contributor to phenotypic innovations in evolutionary history. The diversity of blood oxygen transport traits is the perfect reflection of physiological versatility for evolutionary success among vertebrates. In this study, the evolutionary changes of hemoglobin(Hb) repertoire driven by the recent genome duplications were detected in representative Cyprinidae fish, including eight diploid and four tetraploid species. Comparative genomic analysis revealed a substantial variation in both membership composition and intragenomic organization of Hb genes in these species.Phylogenetic reconstruction analyses were conducted to characterize the evolutionary history of these genes. Data were integrated with the expression profiles of the genes during ontogeny. Our results indicated that genome duplications facilitated the phenotypic diversity of the Hb gene family; each was associated with species-specific changes in gene content via gene loss and fusion after genome duplications. This led to repeated evolutionary transitions in the ontogenic regulation of Hb gene expression.Our results revealed that genome duplications helped to generate phenotypic changes in Cyprinidae Hb systems.     

Stress specific correlated responses in fat content, Hsp70 and dopamine levels in Drosophila melanogaster selected for resistance to environmental stress

Studies of adaptation to stressful environments have frequently encountered cross resistance. This has prompted the hypothesis that certain adaptations confer resistance to multiple stressors. Some of the genes and mechanisms conferring stress resistance have been identified, however, the generality and basis of stress adaptation and cross resistance is still unclear. We investigated several physiological traits that have been previously linked to increased stress resistance: Hsp70 expression, fat content and dopamine levels. Additionally, we studied a behavioural trait, locomotor activity, as a proxy for the physiological state of the organisms. Physiology is the mechanistic link between resistance phenotype and underlying genetic background, and provides insights into the background and generality of cross resistance and correlated responses to selection for stress resistance. We assessed the relationship between the measured traits and stress resistance in a set of lines selected for increased resistance to several environmental stressors. We found that, although all physiological traits displayed significant differentiation among selection regimes, none were consistently associated with increased general stress resistance. This demonstrates that directional changes in Hsp70 expression level, dopamine level and fat content occur in response to the specific requirements of the different stress regimes, rather than as a general response to stress.     

Salt‐tolerance diversity in diploid and polyploid cotton (Gossypium) species

The development of salt‐tolerant genotypes is pivotal for the effective utilization of salinized land and to increase global crop productivity. Several cotton species comprise the most important source of textile fibers globally, and these are increasingly grown on marginal or increasingly saline agroecosystems. The allopolyploid cotton species also provide a model system for polyploid research, of relevance here because polyploidy was suggested to be associated with increased adaptation to stress. To evaluate the genetic variation of salt tolerance among cotton species, 17 diverse accessions of allopolyploid (AD‐genome) and diploid (A‐ and D‐genome) Gossypium were evaluated for a total of 29 morphological and physiological traits associated with salt tolerance. For most morphological and physiological traits, cotton accessions showed highly variable responses to 2 weeks of exposure to moderate (50 mm NaCl) and high (100 mm NaCl) hydroponic salinity treatments. Our results showed that the most salt‐tolerant species were the allopolyploid Gossypium mustelinum from north‐east Brazil, the D‐genome diploid Gossypium klotzschianum from the Galapagos Islands, followed by the A‐genome diploids of Africa and Asia. Generally, A‐genome accessions outperformed D‐genome cottons under salinity conditions. Allopolyploid accessions from either diploid genomic group did not show significant differences in salt tolerance, but they were more similar to one of the two progenitor lineages. Our findings demonstrate that allopolyploidy in itself need not be associated with increased salinity stress tolerance and provide information for using the secondary Gossypium gene pool to breed for improved salt tolerance.     

Adaptation to High Ethanol Reveals Complex Evolutionary Pathways

Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts.     

Pichia stipitis genomics, transcriptomics, and gene clusters

Genome sequencing and subsequent global gene expression studies have advanced our understanding of the lignocellulose-fermenting yeast Pichia stipitis . These studies have provided an insight into its central carbon metabolism, and analysis of its genome has revealed numerous functional gene clusters and tandem repeats. Specialized physiological traits are often the result of several gene products acting together. When coinheritance is necessary for the overall physiological function, recombination and selection favor colocation of these genes in a cluster. These are particularly evident in strongly conserved and idiomatic traits. In some cases, the functional clusters consist of multiple gene families. Phylogenetic analyses of the members in each family show that once formed, functional clusters undergo duplication and differentiation. Genome-wide expression analysis reveals that regulatory patterns of clusters are similar after they have duplicated and that the expression profiles evolve along with functional differentiation of the clusters. Orthologous gene families appear to arise through tandem gene duplication, followed by differentiation in the regulatory and coding regions of the gene. Genome-wide expression analysis combined with cross-species comparisons of functional gene clusters should reveal many more aspects of eukaryotic physiology.     

Evolutionary adaptation to environmental stressors: a common response at the proteomic level

Mechanistic trade‐offs between traits under selection can shape and constrain evolutionary adaptation to environmental stressors. However, our knowledge of the quantitative and qualitative overlap in the molecular machinery among stress tolerance traits is highly restricted by the challenges of comparing and interpreting data between separate studies and laboratories, as well as to extrapolating between different levels of biological organization. We investigated the expression of the constitutive proteome (833 proteins) of 35 Drosophila melanogaster replicate populations artificially selected for increased resistance to six different environmental stressors. The evolved proteomes were significantly differentiated from replicated control lines. A targeted analysis of the constitutive proteomes revealed a regime‐specific selection response among heat‐shock proteins, which provides evidence that selection also adjusts the constitutive expression of these molecular chaperones. Although the selection response in some proteins was regime specific, the results were dominated by evidence for a “common stress response.” With the exception of high temperature survival, we found no evidence for negative correlations between environmental stress resistance traits, meaning that evolutionary adaptation is not constrained by mechanistic trade‐offs in regulation of functional important proteins. Instead, standing genetic variation and genetic trade‐offs outside regulatory domains likely constrain the evolutionary responses in natural populations.