Stomatal versus non-stomatal limitation in salt-stressed tomato: reading gas exchange against fluorescence
Two tomato cultivars are compared in a greenhouse 30 days after transplanting under control (EC 1.2 dS m⁻¹) and saline (EC 6.0 dS m⁻¹, applied as NaCl/CaCl₂) irrigation, four replicates. On the youngest fully expanded leaf the team measures light-saturated net photosynthesis (A, µmol CO₂ m⁻² s⁻¹), stomatal conductance (gs, mol m⁻² s⁻¹), intercellular CO₂ (Ci, µmol mol⁻¹; chamber Ca = 400), dark-adapted Fv/Fm, and leaf N (%). Cultivar A: A 22.4 → 12.1; gs 0.42 → 0.11; Ci 310 → 165; Fv/Fm 0.80 → 0.63; N 3.4 → 3.2. Cultivar B: A 20.8 → 17.9; gs 0.40 → 0.22; Ci 305 → 240; Fv/Fm 0.79 → 0.74; N 3.3 → 3.3. Leaf Na⁺ rose from 15 to 210 µmol g⁻¹ DW in A and from 14 to 95 µmol g⁻¹ DW in B. When a subset of A leaves was re-measured at 800 µmol mol⁻¹ CO₂, A recovered from 12.1 to 18.6.
1. For each cultivar, decide how much of the photosynthetic decline is stomatal and how much is non-stomatal, and justify the conclusion from the data given.
Read the worked answer
Use Ci/Ca as a first discriminator, then look for independent evidence, because a low Ci does not by itself prove stomatal limitation - it can equally reflect low mesophyll conductance.
Cultivar A: Ci/Ca falls from 310/400 = 0.78 to 165/400 = 0.41. A large drawdown of Ci is the classic signature of stomatal closure outrunning the decline in assimilation, and gs fell by 74%. But three observations demand a non-stomatal component:
- Fv/Fm = 0.63, well below the ~0.78–0.83 healthy value, indicating sustained photoinhibition.
- Raising CO₂ to 800 µmol mol⁻¹ restored A only to 18.6, i.e. 83% of the control. If the limitation were purely stomatal, saturating CO₂ should have restored A close to the control value. The residual ~17% is metabolic or mesophyll-resistance limitation.
- Leaf N is essentially unchanged, so the machinery is still there; the impairment is functional, not a loss of protein.
Verdict for A: mixed limitation, with stomatal closure dominant at ambient CO₂ plus a real photochemical/metabolic component.
Cultivar B: Ci/Ca = 0.61 versus 0.76 in its control, gs halved, Fv/Fm only 0.79 → 0.74. Here the decline is predominantly stomatal with only minor photochemical damage. B is the more salt-tolerant genotype on both the gas-exchange and the Na⁺ data (95 versus 210 µmol g⁻¹ DW).
2. The breeder proposes using Fv/Fm at 30 days as the primary selection trait for salt tolerance. Critique that proposal.
Read the worked answer
It is a weak choice for four reasons.
- Discriminating power. In the tolerant cultivar, Fv/Fm falls only from 0.79 to 0.74 - a 0.05 difference. Healthy, unstressed tomato routinely varies by 0.02–0.03 between leaves and between measurement occasions, and dark adaptation for 20–30 min per leaf makes this expensive at breeding scale. You would be selecting within measurement noise.
- It is a symptom, not a mechanism. Fv/Fm reports the fate of absorbed light in photosystem II. It does not measure growth, fruit set, or yield, and a genotype that maintains Fv/Fm by dissipating excess light or by simply growing more slowly is not necessarily tolerant.
- Genotype × environment. Salinity in the field fluctuates with irrigation and evaporation; single-time-point fluorescence in a greenhouse is a poor predictor of season-long performance. Heritability measured this way will be low.
- Better alternatives exist in the very data set. Leaf Na⁺ separates the cultivars two-fold (210 versus 95 µmol g⁻¹ DW) with a plausible mechanism (Na⁺ exclusion) and is cheap and quantitative. Na⁺/K⁺ ratio, yield stability across salinity levels, or Vcmax and Jmax derived from A-Ci curves would all give a stronger and more heritable selection criterion.
Appropriate use of Fv/Fm: as a rapid screen to discard clearly damaged material, and as a supporting line of evidence, not as the primary selection trait.
3. Cultivar A's leaf nitrogen is unchanged while light-saturated photosynthesis has roughly halved. What does this imply mechanistically, and what experiment would separate a diffusional from a metabolic limitation?
Read the worked answer
Leaf N is dominated by Rubisco and thylakoid proteins, so unchanged N with halved A means the photosynthetic apparatus is present but operating below capacity. The lesion is therefore in the operation of the machinery - a regulatory or ionic effect - rather than in protein degradation or remobilisation.
Likely candidates in a leaf carrying 210 µmol g⁻¹ DW Na⁺:
- K⁺ displacement and a high Na⁺/K⁺ ratio in the cytosol and chloroplast, impairing enzyme function.
- Reduced mesophyll conductance (gm), so that the CO₂ concentration at the carboxylation site (Cc) is much lower than the measured Ci. This is the diffusional explanation and it would mimic stomatal limitation in the Ci/Ca analysis.
- Impaired ATP synthesis or electron transport downstream of PSII, consistent with the depressed Fv/Fm, which would lower the RuBP regeneration capacity (Jmax) before it lowers carboxylation capacity (Vcmax).
Design to separate them:
- Simultaneous gas exchange and chlorophyll fluorescence to derive gm by the variable-J method, and construct the A versus Cc curve rather than the A versus Ci curve. If the A-Cc curves of stressed and control leaves superimpose, the limitation is diffusional; if they separate, it is metabolic.
- A-Ci curves at several CO₂ levels to extract Vcmax and Jmax. A fall in both with unchanged N suggests reduced specific activity of Rubisco and of electron transport.
- Measure Rubisco activation state and extractable activity per unit protein, plus leaf K⁺ and the Na⁺/K⁺ ratio, to test the ionic-toxicity hypothesis directly.