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

Intron-mediated induction of phenotypic heterogeneity

Intragenic regions that are removed during maturation of the RNA transcript—introns—are universally present in the nuclear genomes of eukaryotes1. The budding yeast, an otherwise intron-poor species, preserves two sets of ribosomal protein genes that differ primarily in their introns2,3. Although studies have shed light on the role of ribosomal protein introns under stress and starvation4,5,6, understanding the contribution of introns to ribosome regulation remains challenging. Here, by combining isogrowth profiling7 with single-cell protein measurements8, we show that introns can mediate inducible phenotypic heterogeneity that confers a clear fitness advantage. Osmotic stress leads to bimodal expression of the small ribosomal subunit protein Rps22B, which is mediated by an intron in the 5′ untranslated region of its transcript. The two resulting yeast subpopulations differ in their ability to cope with starvation. Low levels of Rps22B protein result in prolonged survival under sustained starvation, whereas high levels of Rps22B enable cells to grow faster after transient starvation. Furthermore, yeasts growing at high concentrations of sugar, similar to those in ripe grapes, exhibit bimodal expression of Rps22B when approaching the stationary phase. Differential intron-mediated regulation of ribosomal protein genes thus provides a way to diversify the population when starvation threatens in natural environments. Our findings reveal a role for introns in inducing phenotypic heterogeneity in changing environments, and suggest that duplicated ribosomal protein genes in yeast contribute to resolving the evolutionary conflict between precise expression control and environmental responsiveness9

TAMAÑO DE NEOBUXBAUMIA TETETZO Y LONGITUD DE SUS ESPINAS APICALES EN UN GRADIENTE DE LUZ BAJO MIMOSA LUISANA, UN ARBUSTO NODRIZA

Environmental conditions and resource availability under the canopy of nurse shrubs in xeric ecosystems are not homogeneous and affect growth rate, survivorship and some morphological traits of young cacti plants. We studied the effect of a gradient of photosynthetically active radiation (PAR) on two traits of the columnar cactus Neobuxbaumia tetetzo: (1) individuals height and (2) length of its apical thorns under the nurse shrub Mimosa luisana in the Zapotitlán Salinas Valley, Puebla (Mexico). We found a positive correlation between the length of the apical thorns and the distance from the nurse shrub, as well as between the height of N. tetetzo and the length of apical thorns. Likewise, the correlation between N. tetetzo size and the distance from the nurse shrub was positive. A multiple regression analysis showed that the length of the apical thorns depends on the size of N. tetetzo individuals, but not on the distance from the nurse shrub. Additionally, the sizes of the N. tetetzo individuals were also dependent on distance from the nurse shrub. When N. tetetzo establishes on the periphery of a nurse shrub the recruitment probability is low as a cause of the high radiation level. Likewise, this factor can have a negative effect on the growth rate because high temperatures reduce photosynthetic rates.Las condiciones ambientales y los recursos bajo el dosel de un arbusto nodriza no son uniformes y afectan las tasas de crecimiento y de supervivencia de las plantas que lo usan como refugio, así como algunas características morfológicas. En el Valle de Zapotitlán Salinas, Puebla, se estudió el efecto que tiene la radiación fotosintéticamente activa (RFA) en función de su distancia al centro de Mimosa luisana sobre dos características morfométricas de Neobuxbaumia tetetzo: (1) la longitud de las espinas apicales y (2) el tamaño de los individuos. Se encontraron correlaciones significativas y positivas entre la longitud máxima de las espinas apicales y su distancia al centro del arbusto, así como entre el tamaño de N. tetetzo y la longitud de las espinas apicales. Del mismo modo, el tamaño de N. tetetzo y su distancia al centro de la nodriza guardan una correlación significativa y positiva. Un análisis de regresión múltiple mostró que el largo de las espinas apicales de N. tetetzo se vincula con el tamaño del individuo, pero no de su distancia al arbusto. Por su parte, el tamaño de N. tetetzo es una característica dependiente de su distancia a la nodriza. Se discute el hecho de que cuando N. tetetzo se establece cerca de la periferia de M. luisana existe una baja probabilidad de reclutamiento debida a la incidencia de altos niveles de radiación, los cuales también pueden afectar negativamente el crecimiento de N. tetetzo, debido a las altas temperaturas asociadas que afectan negativamente sus tasas fotosintéticas

Tamaño de Neobuxbaumia tetetzo y longitud de sus espinas apicales en un gradiente de luz bajo Mimosa luisana, un arbusto nodriza

Las condiciones ambientales y los recursos bajo el dosel de un arbusto nodriza no son uniformes y afectan las tasas de crecimiento y de supervivencia de las plantas que lo usan como refugio, así como algunas características morfológicas. En el Valle de Zapotitlán Salinas, Puebla, se estudió el efecto que tiene la radiación fotosintéticamente activa (RFA) en función de su distancia al centro de Mimosa luisana sobre dos características morfométricas de Neobuxbaumia tetetzo: (1) la longitud de las espinas apicales y (2) el tamaño de los individuos. Se encontraron correlaciones significativas y positivas entre la longitud máxima de las espinas apicales y su distancia al centro del arbusto, así como entre el tamaño de N. tetetzo y la longitud de las espinas apicales. Del mismo modo, el tamaño de N. tetetzo y su distancia al centro de la nodriza guardan una correlación significativa y positiva. Un análisis de regresión múltiple mostró que el largo de las espinas apicales de N. tetetzo se vincula con el tamaño del individuo, pero no de su distancia al arbusto. Por su parte, el tamaño de N. tetetzo es una característica dependiente de su distancia a la nodriza. Se discute el hecho de que cuando N. tetetzo se establece cerca de la periferia de M. luisana existe una baja probabilidad de reclutamiento debida a la incidencia de altos niveles de radiación, los cuales también pueden afectar negativamente el crecimiento de N. tetetzo, debido a las altas temperaturas asociadas que afectan negativamente sus tasas fotosintéticasEnvironmental conditions and resource availability under the canopy of nurse shrubs in xeric ecosystems are not homogeneous and affect growth rate, survivorship and some morphological traits of young cacti plants. We studied the effect of a gradient of photosynthetically active radiation (PAR) on two traits of the columnar cactus Neobuxbaumia tetetzo: (1) individuals height and (2) length of its apical thorns under the nurse shrub Mimosa luisana in the Zapotitlán Salinas Valley, Puebla (Mexico). We found a positive correlation between the length of the apical thorns and the distance from the nurse shrub, as well as between the height of N. tetetzo and the length of apical thorns. Likewise, the correlation between N. tetetzo size and the distance from the nurse shrub was positive. A multiple regression analysis showed that the length of the apical thorns depends on the size of N. tetetzo individuals, but not on the distance from the nurse shrub. Additionally, the sizes of the N. tetetzo individuals were also dependent on distance from the nurse shrub. When N. tetetzo establishes on the periphery of a nurse shrub the recruitment probability is low as a cause of the high radiation level. Likewise, this factor can have a negative effect on the growth rate because high temperatures reduce photosynthetic rate

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.status: publishe

Dynamics and linkage of mutations in evolved populations of reactor 2.

<p>Mutations (reaching a frequency of least 20% in the evolved population samples) and corresponding frequencies were identified from population sequencing data. Muller diagram represents the hierarchical clustering of these mutations, with each color block representing a specific group of linked mutations. Indels are designated with ^I, whereas heterozygous mutations are in italics. Mutations present as heterozygous mutations in all clones of a specific time point and present at a frequency of 50% in the population, are depicted as a frequency of 100% in the population, since it is expected that all cells in the population contain this mutation. After 80 generations, a mutator phenotype appeared in this reactor (indicated by arrow under graph), which coincides with the rise in frequency of an indel in the mismatch repair gene <i>MSH2</i>. Frequencies of haplotypes can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005635#pgen.1005635.s026" target="_blank">S2 Table</a>. Dynamics and linkage for reactor 1 is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005635#pgen.1005635.s007" target="_blank">S7 Fig</a>.</p

Adaptive pathways are involved in cell cycle, DNA repair and protoporphyrinogen metabolism.

<p>Shown is the sub-network that prioritizes putative adaptive mutations by applying PheNetic on all selected mutations, excluding those originating from the populations with a mutator phenotype i.e. reactor 2 and 6. The nodes in the network correspond to genes and/or their associated gene products. Node borders are colored according to the reactors containing the populations in which these genes were mutated. Nodes are colored according to gene function, for each gene the most enriched term is visualized (grey indicates no enrichment). Cell cycle related processes have been subdivided into DNA replication and interphase. The edge colors indicate different interaction types. Orange lines represent metabolic interactions, green lines represent protein-protein interactions, red lines represent protein-DNA interactions. Sub-networks extracted by separately analyzing the mutated genes observed in each of the different populations (reactors) are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005635#pgen.1005635.s008" target="_blank">S8</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005635#pgen.1005635.s013" target="_blank">S13</a> Figs.</p