describe the role of abscisic acid in the closure of stomata during times of water stress, including the role of calcium ions as a second messenger

Homeostasis in Plants – Syllabus 14 (Cambridge 9700)

14.1 Plant Water Relations & Water Potential

Water moves in plants according to the total water potential (Ψ), the sum of its solute (Ψ_s) and pressure (Ψ_p) components:

\$\Psi = \Psis + \Psip\$

• Ψ_s (solute potential) – always negative; expressed as

  \$\Psi_s = -\,i C R T\$

  where i = ionisation constant, C = molar concentration, R = 0.00831 L MPa mol⁻¹ K⁻¹, T = temperature (K).

• Ψ_p (pressure potential) – positive when turgor is present; zero in dead tissue or the xylem.

Example calculation (AO2 style):

For a 0.10 M sucrose solution at 25 °C (298 K) with i = 1:

\$\Psi_s = - (1)(0.10\ \text{mol L}^{-1})(0.00831\ \text{MPa L mol}^{-1}\text{K}^{-1})(298\ \text{K}) \approx -0.25\ \text{MPa}\$

Link to guard‑cell turgor: When soil water potential (Ψ_soil) falls, water moves out of the leaf, making leaf Ψ less negative (i.e., closer to zero). The resulting fall in Ψ_p in guard cells reduces their turgor, causing stomatal closure – a direct application of the Ψ equation in exam questions.

14.2 Hormonal Control of Stomatal Aperture

Abscisic Acid (ABA) – the primary drought signal

• Water deficit → ↑ ABA synthesis in mesophyll chloroplasts.
• ABA diffuses through the apoplast to guard cells.
• In guard cells, ABA binds PYR/PYL/RCAR receptors, inhibiting PP2C phosphatases and allowing SnRK2 kinases to become active.
• Active SnRK2 phosphorylates Ca²⁺ channels (causing a Ca²⁺ influx) and SLAC1 anion channels (promoting Cl⁻/NO₃⁻ efflux).
• Elevated cytosolic Ca²⁺ acts as a second messenger (see 14.5).
• Ca²⁺‑dependent protein kinases (CDPKs) and calmodulin activate outward‑rectifying K⁺ channels (GORK) → K⁺ loss.
• Loss of anions, K⁺ and organic acids lowers guard‑cell Ψ_s, water exits the cell, Ψ_p falls and the stomatal pore closes.

Other hormones – contrasting mechanisms

During drought, the rapid rise in ABA overrides the opening signals from auxin, cytokinin or low ethylene, making ABA the dominant regulator (AO2).

14.3 Abiotic Factors that Modulate Stomata

• Light (blue‑light receptors – phototropins) – Phototropin activation stimulates plasma‑membrane H⁺‑ATPases, creating an electrochemical gradient that drives K⁺ uptake through inward‑rectifying channels, leading to guard‑cell swelling and stomatal opening (required syllabus point).
• CO₂ concentration – High internal CO₂ raises cytosolic HCO₃⁻, which activates SLAC1‑like anion channels, causing depolarisation and stomatal closure.
• Relative humidity / vapour pressure deficit (VPD) –

  \$\text{VPD}=es - ea\$

  where e_s = saturation vapour pressure (function of temperature) and e_a = actual vapour pressure.

  High VPD → rapid transpiration → leaf Ψ becomes less negative → ABA synthesis increases, reinforcing closure.

• Temperature – Raises VPD and can directly stimulate ABA biosynthesis; extreme heat also affects membrane fluidity and ion‑channel kinetics.

14.4 ABA‑Induced Stomatal Closure – Signal Transduction Pathway

1. Water deficit → ↑ ABA synthesis in mesophyll chloroplasts.
2. ABA moves apoplastically to guard cells.
3. ABA binds PYR/PYL/RCAR receptors on the guard‑cell plasma membrane.
4. Receptor activation inhibits PP2C phosphatases → SnRK2 kinases become active.
5. SnRK2 phosphorylates:

   • Plasma‑membrane Ca²⁺ channels → rapid Ca²⁺ influx.
   • SLAC1 anion channels → Cl⁻/NO₃⁻ efflux.

6. Elevated cytosolic Ca²⁺ acts as a second messenger (see 14.5).
7. Ca²⁺‑dependent protein kinases (CDPKs) and calmodulin activate outward‑rectifying K⁺ channels (GORK) → K⁺ loss.
8. Combined loss of anions, K⁺ and organic acids reduces guard‑cell Ψ_s (becomes less negative).
9. Water exits the guard cells, Ψ_p falls, and the stomatal pore closes.

14.5 Calcium Ions as a Second Messenger

Ca²⁺ links ABA perception to ion‑channel regulation. Its functions are:

• Signal amplification – Transient Ca²⁺ spikes open CDPKs and bind calmodulin, which phosphorylate multiple downstream targets.
• Channel regulation – CDPK‑phosphorylated SLAC1 enhances anion efflux; GORK activation drives K⁺ efflux.
• Crosstalk with reactive oxygen species (ROS) – Ca²⁺ activates NADPH oxidases (RBOH), generating ROS that further modulate SLAC1 and GORK activity.

14.6 Key Molecular Players

14.7 Integration with Whole‑Plant Processes (Learning‑Outcome Links)

• Transport in Plants (Topic 7) – Stomatal closure reduces transpiration, helping maintain a favourable water‑potential gradient (soil → root → xylem → leaf) and protecting the cohesion‑tension mechanism (AO1, AO2).
• Gas Exchange (Topic 12) – Closure limits CO₂ uptake, directly lowering the substrate for the Calvin cycle (AO1) and reducing O₂ loss (AO2).
• Photosynthesis (Topic 13) – Decreased internal CO₂ reduces Rubisco carboxylation rate, illustrating the trade‑off between water conservation and carbon gain (AO2).
• Respiration (Topic 14) – Lower O₂ availability can slightly affect mitochondrial respiration in leaf tissue (AO3).

14.8 Suggested Practical Investigation – Effect of Exogenous ABA on Stomatal Aperture

Objective (AO2): Quantify how 10 µM ABA influences stomatal pore size in a fast‑growing dicot.

1. Collect fresh leaf epidermal peels from Phaseolus vulgaris (or another herbaceous dicot).
2. Prepare three treatment groups (minimum n = 5 peels per group):

   • Control – distilled water.
   • ABA – 10 µM ABA solution.
   • Mock – solvent control (e.g., 0.01 % ethanol) to check for solvent effects.

3. Place each peel in a slide chamber with the appropriate solution, keep under constant cool white light (≈150 µmol m⁻² s⁻¹) for 30 min. Include a dark‑incubated set (no light) to test the interaction of light and ABA (AO3).
4. Capture images with a compound microscope (×400) and measure pore width and length using ImageJ or a calibrated ocular micrometer.
5. Calculate % change in aperture relative to the control:

   \$\%\,\text{change}= \frac{(\text{Aperture}{\text{treatment}}-\text{Aperture}{\text{control}})}{\text{Aperture}_{\text{control}}}\times100\$

6. Analyse data with a t‑test (or ANOVA if more than two groups) to assess statistical significance (AO3).

Assessment objectives addressed: AO2 – interpretation of quantitative data; AO3 – design, use of controls, statistical analysis, evaluation of experimental limitations (e.g., peel age, light intensity variation).

14.9 Key Points for Revision (AO1/AO2)

• Water stress → ↑ ABA synthesis in mesophyll chloroplasts.
• ABA binds PYR/PYL/RCAR → PP2C inhibition → SnRK2 activation.
• SnRK2 phosphorylates Ca²⁺ channels → rapid rise in cytosolic Ca²⁺.
• Ca²⁺ (second messenger) activates CDPKs, calmodulin and ROS production, which together open SLAC1 (anion) and GORK (K⁺) channels.
• Efflux of Cl⁻, NO₃⁻, K⁺ and organic acids lowers guard‑cell Ψ_s; water follows, Ψ_p falls.
• Reduced guard‑cell turgor closes the stomatal pore, conserving water and maintaining hydraulic integrity.
• Auxin, cytokinin and low ethylene promote opening by stimulating H⁺‑ATPase activity and K⁺ uptake – opposite to ABA’s effect.
• Blue‑light receptors → H⁺‑ATPase activation → K⁺ influx → opening.
• High internal CO₂ → HCO₃⁻‑mediated activation of anion channels → closure.
• High VPD (VPD = e_s − e_a) raises transpiration, lowers leaf Ψ, stimulates ABA synthesis.
• Stomatal behaviour is tightly linked to whole‑plant water transport, gas exchange, photosynthetic carbon gain and respiratory O₂ supply.