A comparative study on physiological responses to drought in wild Vitis species

Context and purpose of the study:Crossings of three wild Vitis species are commonly used worldwide as rootstocks in grape production. Disease resistance and vigor are among the most important factors for their selection. With climate change resulting in increasing water limitations, finding rootstocks conferring increased tolerance to drought will be of great importance as well. Therefore, identifying Vitis species with improved drought tolerance, and incorporating them into breeding programs could contribute to more resilient rootstocks under water-limiting conditions. Furthermore, these species will serve as valuable resources for increasing the genetic variation of the current rootstocks available. Investigating the leaf physiology of these species is pivotal, potentially offering valuable insights into their adaptive mechanisms under drought stress. We hypothesized that species native to drier habitats would exhibit a superior physiological performance under drought stress. Materials and methods: The root and canopy physiological characteristics, and the anatomical and biochemical bases of photosynthetic capacity of nine North American wild Vitis species across a wide latitudinal range (New England through Mexico) under two soil moisture treatments (controlled dry down (20–40% w/w ‘drought’) and maintained irrigated (70–90% w/w ‘control’)), were evaluated using a whole-plant experimental approach. We investigated the links between leaf structural diversity and physiological features that enhance photosynthetic capacity under controlled, non-stressed conditions and whether these relationships are upheld under prolonged water stress. Experiments were performed in a greenhouse under ambient atmospheric conditions using clonal and non-grafted saplings. Physiological parameters measured throughout the experiment included midday and predawn leaf water potentials, leaf gas exchange, root and leaf biomass, and spectral measurements. Additionally, X-ray imaging of plant tissues was performed at a single time point mid-experiment, and manual segmentation was used to prepare images for auto-segmentation using machine learning algorithms. Linear regression models were used to describe the relationships between anatomical and physiological variables, and their associations with biogeoclimatic variables. Results and discussion: Our data shows the impact of drought treatment and indicated differential responses to drought stress across species. Furthermore, structural differences that drive photosynthetic responses were observed. Elucidating canopy traits associated with improved performance under drought conditions could facilitate the rapid screening of germplasms to develop drought- tolerant rootstocks in the future. Keywords: Vitis, grapevines, water-use efficiency, water stress, drought toleranc

Assembling linear DNA templates for in vitro transcription and translation.

Cell-free expression systems provide straightforward access from genes to the corresponding proteins, involving fewer handling steps than in vivo procedures. A quick procedure to assemble a gene of interest into a linear DNA template together with 3'- and 5'-untranslated regions using a coupled uracil-excision-ligation strategy based on USER Enzyme and T4 DNA ligase. This methodology will be useful for repeated cycles of expression and in vitro selection, in which gene libraries are repeatedly assembled and their products and templates regenerated

Droplets as reaction compartments for protein nanotechnology

Extreme miniaturization of biological and chemical reactions in pico- to nanoliter microdroplets is emerging as an experimental paradigm that enables more experiments to be carried out with much lower sample consumption, paving the way for high-throughput experiments. This review provides the protein scientist with an experimental framework for (a) formation of polydisperse droplets by emulsification or, alternatively, of monodisperse droplets using microfluidic devices; (b) construction of experimental rigs and microfluidic chips for this purpose; and (c) handling and analysis of droplets

Reverse evolution leads to genotypic incompatibility despite functional and active site convergence

Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>104-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peak

Reverse evolution leads to genotypic incompatibility despite functional and active site convergence.

Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>10(4)-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peaks

Functional analysis of a random mutant library.

<p>(A) Changes in phosphotriesterase (native; PTE) and arylesterase (promiscuous; AE) activities among variants from a trinucleotide substitution library. The enzymatic activities for each variant (shown as grey dots) are plotted relative to those of <i>wt</i>PTE. Data are averages of triplicate values from three independent experiments and error bars represent +/- 1 SEM. (B) Distribution of the mutational impact on phosphotriesterase and arylesterase activities. Mutations are classified as strongly deleterious (>2-fold activity decrease relative to <i>wt</i>PTE), weakly deleterious (2-fold—1.3-fold decrease), neutral (<1.3-fold change), and positive (>1.3-fold increase). Frequencies are indicated with their corresponding 95% confidence intervals.</p

Functional trade-offs in protein evolution.

<p>Strong trade-offs result when mutations increasing the new function have a large effect on the original function. When the effect on the original function is mild, trade-offs are weak. Weak trade-offs channel evolution through a generalist regime where the enzyme catalyzes both reactions with high efficiency.</p

Evolution from AtzA to TriA (adapted from reference [34]).

<p>(A) AtzA catalyzes the dechlorination of atrazine (AtzA activity). TriA catalyzes the deamination of melamine (TriA activity). TriA catalyzed the dechlorination reaction promiscuously. Deamination by AtzA could not be detected. (B) A possible uphill evolutionary trajectory from AtzA to TriA determined by Noor et al. In each round of evolution, a single point mutation was added in the order shown in (C)—(F) (see also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006305#pgen.1006305.s008" target="_blank">S8 Table</a></b>). (C)—(F) Effect of all single point mutations separating AtzA and TriA (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006305#pgen.1006305.s009" target="_blank">S9</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006305#pgen.1006305.s010" target="_blank">S10</a> Tables</b>). (C) Effect of mutations in the AtzA background on AtzA activity and (D) TriA activity. (E) Effect of mutations in the TriA background on TriA activity and (F) AtzA activity. Activities are expressed as <i>k</i>_<i>cat</i>/<i>K</i>_<i>M</i> values. Relative activities could not be calculated because several variants do not have detectable activity. Amino acids found in AtzA are shown in lower-case italics.</p

Distribution of mutational effects in the evolution of PTE and AtzA.

<p>Distribution of mutational effects in the evolution of PTE and AtzA.</p

Effect of all single point mutations obtained over the evolution.

<p>(A)-(C) Effect of mutations on PTE activity (A) in the <i>wt</i>PTE background, (B) upon their occurence in the evolution, and (C) in the AE background. *Phosphotriesterase activity was too low to be determined in AE-R254<i>h</i>, but at least 10-fold reduced compared to AE. (D)-(F) Effect of mutations on AE activity (D) in the <i>wt</i>PTE background, (E) upon their occurrence in the evolution, and (F) in the AE background. **Arylesterase activity was reduced to 1.9×10⁻⁵ times the level of AE and is therefore not shown to scale. Activities are given relative to the respective parent background. Mutations causing a >1.3-fold change compared to the parent mutant (dotted line) are considered non-neutral. A student T-test was performed and p-values compared to each parent were calculated (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006305#pgen.1006305.s002" target="_blank">S2</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006305#pgen.1006305.s004" target="_blank">S4</a> Tables</b>). The 1.3-fold effect of T341<i>i</i> on AE activity in the AE background as well as the effect of <i>t</i>199I, <i>l</i>140M and <i>t</i>45A on PTE activity in the evolution is statistically not significant. Note that in the evolution, <i>f</i>306 was first mutated to L and then to I and therefore, the direct effect of <i>f</i>306I could not be determined. Amino acids found in <i>wt</i>PTE are shown in lower-case italics.</p