In the budding yeast Saccharomyces cerevisiae, the TOR (Target of Rapamycin) signaling pathway coordinates cell growth with nutrient availability. When amino acids are abundant, active TORC1 (containing the kinase Tor1) inhibits the Sit4 phosphatase (via the Tap42 regulator); as a result, the transcriptional regulator Gln3 remains phosphorylated and is held in the cytoplasm (bound to 14-3-3 proteins), preventing transcription of nitrogen-catabolite-repressed (NCR) genes such as GAP1 (the general amino-acid permease). When amino acids are limiting, TORC1 is inactive, Sit4 inhibition is released, and active Sit4 dephosphorylates Gln3; dephosphorylated Gln3 then enters the nucleus and activates GAP1 transcription. A simplified model of this pathway is shown in Figure 1.

To dissect the roles of these components, researchers used a prototrophic wild-type strain of S. cerevisiae (BY4741 background) to construct two single-gene-deletion strains: gln3Δ (Gln3 knockout) and sit4Δ (Sit4 knockout). They grew each strain in synthetic complete medium (SC, amino acids replete) and in synthetic minimal medium with only a poor nitrogen source (proline; amino acids limiting). They measured the specific activity of the Gap1 permease (¹⁴C-citrulline uptake, nmol·min⁻¹·OD₆₀₀⁻¹) and the relative level of GAP1 mRNA by RT-qPCR, with wild-type cells in SC medium set to 1.0 (Table 1).

Table 1. Gap1 Uptake Activity and Relative GAP1 mRNA in Wild-Type, gln3Δ, and sit4Δ Strains of S. cerevisiae in Replete (SC) and Nitrogen-Limited (Proline) Media

(a) Describe how phosphorylation of a transcriptional regulator such as Gln3 can prevent it from activating its target gene. Explain how a signal can be amplified during transduction in a kinase cascade such as the TOR pathway.

(b) Based on Table 1, identify a dependent variable in the researchers’ experiment. Justify the researchers’ use of the same BY4741 parental strain to construct both deletion mutants. Justifythe researchers’ decision to delete only a single gene in each mutant strain rather than combining deletions.

(c) Based on Table 1, identify the strain and medium that produce the highest relative amount of GAP1 mRNA. Calculate the fold change in Gap1 uptake activity in wild-type cells shifted from SC medium to proline medium.

(d) In a follow-up experiment, researchers constructed a tor1Δ strain in which the catalytic kinase subunit of TORC1 is missing. Based on Figure 1, predict the effect of this deletion on GAP1 expression when the strain is grown in SC medium (amino acids replete). Provide reasoning to justify your prediction.

Submerged aquatic plants adjust their photosynthetic machinery to the light environment of their habitat. To investigate how short-term acclimation to irradiance affects net photosynthesis, scientists grew individual shoots of six freshwater macrophyte species for 14 days in one of two light treatments: low light (100 μmol photons·m⁻²·s⁻¹, typical of turbid water) or high light (800 μmol photons·m⁻²·s⁻¹, typical of clear shallow water). They then measured net O₂ evolution (μmol O₂·g⁻¹ fresh mass·h⁻¹) of excised leaves at a common assay irradiance of 400 μmol photons·m⁻²·s⁻¹. Values are means ± 2 SE, n = 6 plants per species per treatment (Table 1).

Table 1. Net O₂ Evolution in Six Freshwater Macrophyte Species after 14-Day Acclimation to Low Light (100 μmol·m⁻²·s⁻¹) or High Light (800 μmol·m⁻²·s⁻¹)

(a) Describe the role of the thylakoid membrane in the light-dependent reactions of photosynthesis.

(b) Using the data in Table 1: (i) identify the independent and dependent variables and state the axis labels (with units) you would use to display the data; (ii) describe the qualitative shape of the relationship between acclimation irradiance and net O₂ evolution across the six species; (iii) cite one specific numerical data point from Table 1 (species, treatment, and value) that supports your description; and (iv) determine which species show a difference in net O₂ evolution between low- and high-light acclimation.

(c) Based on the data in Table 1, describe the relationship between acclimation light intensity and net O₂ evolution in these macrophytes.

(d) A researcher identifies an individual Elodea canadensis plant whose leaves are pale green rather than the typical dark green. The phenotype results from a mutation in a chloroplast-genome-encoded core subunit of photosystem II (PSII) that prevents proper assembly of the PSII reaction-center complex. Because affected leaves cannot sustain photosynthetic electron transport, chlorophyll bound to the non-functional PSII complexes is degraded through photooxidative stress, leaving the leaves pale and chlorotic. Elodea reproduces sexually, and the pale individual is a female (ovule-producing) plant. Scientists cross the pale female with a dark-green male. Predict the leaf-color phenotype(s) of the F₁ offspring. Provide reasoning to justify your prediction. Explain why genetically identical Elodea clones can differ in the density or size of chloroplasts in their leaves when grown in different light environments.