1 Experimental design

The results presented in this document are based on three independent experiments. All three revealed a “stay-green” phenotype, but only under sulfur-deficient conditions and exclusively in a recently characterized mutant of a sulfate transporter: the pea PsSULTR4 mutant.

Pea plants were grown in pots containing a mixture of perlite/sand (2/1, v:v), in a greenhouse, at a day/night temperature of 18/14°C, with supplemental artificial lighting (16h photoperiod, 250 μmol m^–2 s^–1). The pots were irrigated with a nitrate- and S-rich nutrient solution containing 0.3 mM MgSO₄ (S sufficiency, +S conditions) as described in (Zuber et al. 2013). At a late vegetative stage (about 5 days before flowering, 8th node stage, 3 weeks-old plants), half of the plants from each genotype were subjected to S deficiency (–S) by rinsing the substrate twice with deionized water, and twice with the –S solution in which MgSO₄ has been replaced by MgCl₂. In all –S experiments, S was deprived up to maturity of the plants. For the third experiment, only the –S condition was applied.

Experiment 1 – Transcriptomic profiling:

A microarray analysis was conducted to assess the transcriptional activity in the leaves of each genotype.
This approach aimed to identify differentially expressed genes in response to sulfur deficiency.
The results provided a first insight into the molecular response of the sultr4 mutant compared to the wild-type under sulfur-limited conditions.

Experiment 2 – Physiological and metabolic measurements:

This experiment focused on physiological traits and metabolite concentrations in both wild-type and mutant plants.
Measurements were taken under both sulfur-sufficient and sulfur-deficient conditions.
The data helped identify specific metabolic shifts associated with the stay-green phenotype in the mutant line.

Experiment 3 – Focused study under sulfur stress conditions:

This experiment exclusively targeted sulfur-deficient conditions to further investigate the delay in senescence observed in the sultr4 mutant.
We performed shoot-debudding (removal of reproductive sinks) to test two alternative hypotheses: whether the delayed senescence is due to impaired vacuolar sulfate remobilization, or simply a consequence of reduced sink strength (fewer seeds).
If stay-green leaves persist in the mutant but not in the wild-type after debudding, it suggests that sulfate remobilization directly regulates senescence. Conversely, if both mutant and wild-type remain green after debudding, it indicates that the stay-green phenotype is likely driven by the reduced number of seeds.

--- output: html_document editor_options: chunk_output_type: console editor: markdown: wrap: sentence --- ## Experimental design {#experimental_design} The results presented in this document are based on three independent experiments. All three revealed a “stay-green” phenotype, but only under sulfur-deficient conditions and exclusively in a recently characterized mutant of a sulfate transporter: the pea PsSULTR4 mutant. Pea plants were grown in pots containing a mixture of perlite/sand (2/1, v:v), in a greenhouse, at a day/night temperature of 18/14°C, with supplemental artificial lighting (16h photoperiod, 250 μmol m^–2^ s^–1^). The pots were irrigated with a nitrate- and S-rich nutrient solution containing 0.3 mM MgSO~4~ (S sufficiency, +S conditions) as described in [@zuber2013]. At a late vegetative stage (about 5 days before flowering, 8th node stage, 3 weeks-old plants), half of the plants from each genotype were subjected to S deficiency (–S) by rinsing the substrate twice with deionized water, and twice with the –S solution in which MgSO~4~ has been replaced by MgCl~2~. In all –S experiments, S was deprived up to maturity of the plants. For the third experiment, only the –S condition was applied. [**Experiment 1 – Transcriptomic profiling:**](#transcriptomic) - A microarray analysis was conducted to assess the transcriptional activity in the leaves of each genotype. - This approach aimed to identify differentially expressed genes in response to sulfur deficiency. - The results provided a first insight into the molecular response of the sultr4 mutant compared to the wild-type under sulfur-limited conditions. **Experiment 2 – Physiological and metabolic measurements:** - This experiment focused on [physiological traits](#biomass) and [metabolite](#metabolomic) concentrations in both wild-type and mutant plants. - Measurements were taken under both sulfur-sufficient and sulfur-deficient conditions. - The data helped identify specific metabolic shifts associated with the stay-green phenotype in the mutant line. **Experiment 3 – Focused study under sulfur stress conditions:** - This experiment exclusively targeted sulfur-deficient conditions to further investigate the delay in senescence observed in the sultr4 mutant. - We performed shoot-debudding (removal of reproductive sinks) to test two alternative hypotheses: whether the delayed senescence is due to impaired vacuolar sulfate remobilization, or simply a consequence of reduced sink strength (fewer seeds). - If stay-green leaves persist in the mutant but not in the wild-type after debudding, it suggests that sulfate remobilization directly regulates senescence. Conversely, if both mutant and wild-type remain green after debudding, it indicates that the stay-green phenotype is likely driven by the reduced number of seeds.