Water Relations & Hydraulic Architecture

Lab 6: Water Relations & Hydraulic Architecture

Objectives

By the end of this lab, you'll understand:

• How much water plants lose through transpiration

• Which part of the leaf controls water loss (stomata location)

• How plant structure affects water transport efficiency

• How plants respond to water stress (wilting and recovery)

• Why desert plants have specialized water management strategies

• The relationship between plant architecture and hydraulic efficiency

Background

Every plant faces a dilemma: They need to open pores (stomata) to take in CO₂ for photosynthesis, but this causes water loss. Desert plants face an extreme version of this challenge!

Key Concepts:

Transpiration: Water loss through leaf pores (stomata)

• Necessary for cooling and nutrient transport

• Can't be completely stopped without stopping photosynthesis

• Desert plants lose 100-500× their body weight in water per growing season

Hydraulic Architecture: The "plumbing system" moving water from roots to leaves

• Like pipes in a building—diameter and length matter

• Taller plants or plants with more leaves need better plumbing

• Trade-off between safety (narrow vessels resist cavitation) and efficiency (wide vessels move more water)

Water Stress: What happens when water loss exceeds water uptake

• Wilting (loss of turgor pressure)

• Stomatal closure (stops photosynthesis but saves water)

• Leaf rolling or folding (reduces surface area exposed to sun)

Materials

• Clear plastic bags

• Rubber bands or twist ties

• Measuring tape or ruler

• Measuring cup

• Scale

• Timer or smartphone

• Camera

• Petroleum jelly (Vaseline)

• String

• Pruning shears

• Trowel

Part 1: Measure Transpiration Rates

Goal: Quantify how much water plants lose under different environmental conditions. This reveals which factors most strongly affect water loss.

Experiment 1: Basic Transpiration Measurement

The principle: Water evaporating from leaves will condense inside sealed plastic bags. More condensation = more transpiration.

Step 1: Select Your Study Plants (Before 8 AM)

Choose 6 plants that represent different conditions:

Option A: Sunlight comparison

• 3 plants in full sun

• 3 plants in shade

• (Controls for light effects on transpiration)

Option B: Soil moisture comparison

• 3 plants in wet/irrigated soil

• 3 plants in dry soil

• (Controls for water availability effects)

Option C: Mixed design (recommended)

• 2 plants in full sun + dry soil

• 2 plants in full sun + wet soil

• 2 plants in shade + wet soil

This lets you test multiple variables!

Selection criteria:

• Healthy plants (no disease or damage)

• Similar size (makes comparisons easier)

• Accessible leaves (you'll need to reach them)

• Won't be disturbed (away from foot traffic)

Step 2: Bag the Leaves

For each of your 6 plants, you'll bag 3 leaves (18 bags total).

Why 3 leaves per plant? Replication! Individual leaves vary—averaging 3 gives you reliable data.

Detailed procedure:

1. Select appropriate leaves:

   • Mature leaves (fully expanded, not brand new)

   • Healthy appearance (green, no spots or holes)

   • Not touching other leaves (prevents shading of bag)

   • Similar size across all plants (for fair comparison)

2. Prepare the bag:

   • Check for holes (hold up to light)

   • Label with waterproof marker on tape:

     • Plant ID

     • Leaf number (A, B, or C)

     • Condition (e.g., "Full sun - Dry soil")

     • Start time

3. Enclose the leaf:

   • Gently slip bag over leaf (don't bend or damage)

   • Pull bag down to petiole (leaf stalk)

   • Ensure entire leaf blade is inside bag

   • Avoid trapping other leaves or stems

4. Seal around the petiole:

   • Use rubber band or twist tie

   • Seal should be snug but not crushing

   • No air gaps (water vapor must stay in bag)

   • Don't restrict water flow up the petiole

5. Check your setup:

   • Bag inflated slightly (not crumpled on leaf)

   • No holes or tears

   • Label clearly visible

   • Leaf still receiving light appropriate to its location

Troubleshooting:

• If bag keeps sliding off: Use two twist ties

• If leaf is too big: Use gallon-size bag

• If plant has compound leaves: Bag the whole leaflet cluster

Step 3: Record Initial Conditions

For each plant, note:

Environmental data:

• Time bagged: _____ AM

• Air temperature: _____ °F

• Weather:  /  Sunny  /  Partly cloudy  /  Overcast

• Wind:  /  Calm  /  Light breeze  /  Windy

Soil conditions:

• Soil moisture:  /  Dry  /  Moist  /  Wet

• Recent irrigation:  /  Yes (when: _____)  /  No

Light conditions:

• Location:  /  Full sun  /  Partial shade  /  Deep shade

• Estimated light level (use light meter if available): _____ lux

Step 4: Wait 4 Hours

While you wait, work on Parts 2 and 3 of the lab!

During the waiting period:

• Check bags after 1 hour to ensure they're still sealed

• Photograph bags showing condensation developing

• Note any changes in weather

• Complete other lab sections

What's happening inside the bags:

• Stomata on leaves open (especially in sunlight)

• Water vapor escapes from stomata

• Humid air inside bag becomes saturated

• Water condenses on cool bag surface

• Visible droplets form

Expected timeline:

• 30 min: Slight fogging visible

• 1 hour: Clear droplets forming

• 2 hours: Many droplets, bag interior wet

• 4 hours: Heavy condensation (if transpiring actively)

Step 5: Measure Water Accumulation

After exactly 4 hours, quickly measure water collected.

Preparation (do this BEFORE removing bags):

1. Set up workstation in shade

2. Have paper towels ready

3. Pre-weigh dry paper towels:

   • Place paper towel on scale

   • Record weight: _____ g

   • Label paper towel with bag ID

Measurement procedure:

Method 1: Paper towel absorption (most practical)

1. Remove bag carefully (don't spill water)

2. Immediately wipe all water from inside bag onto pre-weighed paper towel

   • Wipe all inner surfaces

   • Squeeze any pooled water into towel

   • Work quickly (water evaporates fast in desert!)

3. Weigh wet paper towel

   • Record weight: _____ g

4. Calculate water collected:

   • Water mass = (wet weight) - (dry weight)

   • Example: 12.5 g - 10.0 g = 2.5 g water

Method 2: Droplet counting (if very little water)

If condensation is minimal:

1. Count visible droplets

2. Estimate size: Small / Medium / Large

3. Rate overall: None / Light / Moderate / Heavy

Method 3: Volume measurement (if lots of water)

If water pools in bag corners:

1. Weigh an empty cup

2. Pour water into cup

3. Weigh cup with water

4. Record volume in mL

5. Convert: 1 mL water ≈ 1 g

Step 6: Calculate Transpiration Rate

For each leaf:

Transpiration rate = Water collected (g) ÷ Time (4 hours)

Example: 2.5 g ÷ 4 hours = 0.625 g/hour

For each plant (average of 3 leaves):

• Leaf A: _____ g/hour

• Leaf B: _____ g/hour

• Leaf C: _____ g/hour

• Plant average: _____ g/hour

Step 7: Record Your Data

Create a complete data table:

Analysis Questions:

1. Which plant had the highest transpiration rate? _____

   • What conditions did it experience? _____

2. Which plant had the lowest transpiration rate? _____

   • What conditions did it experience? _____

3. Compare full sun vs. shade plants:

   • Average rate in sun: _____ g/hr

   • Average rate in shade: _____ g/hr

   • Difference: _____ × higher in sun

4. Compare wet vs. dry soil plants:

   • Average rate in wet soil: _____ g/hr

   • Average rate in dry soil: _____ g/hr

   • Pattern: Plants in wet soil transpire _____ (more/less)

5. Why do these patterns make sense?

   • Sun increases transpiration because: _____

   • Wet soil increases transpiration because: _____

Experiment 2: Which Leaf Surface Releases Water?

Goal: Discover where stomata are located on leaves. Different plants put stomata in different places to manage water loss.

The hypothesis: Most water escapes from the leaf surface with the most stomata. Blocking that surface should reduce transpiration significantly.

Background: Stomata Distribution

Stomata (singular: stoma) are tiny pores that open and close.

Three common patterns:

1. Hypostomatous: Stomata only on bottom (underside)

   • Most common in trees and shrubs

   • Bottom is cooler, more humid (less water loss)

   • Examples: Oak, maple, most broadleaf trees

2. Amphistomatous: Stomata on both top and bottom

   • Common in grasses and crops

   • Allows high photosynthesis rate

   • Examples: Corn, wheat, grasses

3. Epistomatous: Stomata only on top (rare)

   • Seen in floating aquatic plants

   • Examples: Water lily (top must exchange gases with air)

Step 1: Select Plants (Before 8 AM)

Choose 3 different plant types to test different stomata distributions:

Plant Type 1: Grass or grass-like plant (monocot)

• Examples: Lawn grass, fountain grass, ornamental grasses

• Prediction: Stomata on both surfaces (amphistomatous)

Plant Type 2: Plant with compound leaves

• Examples: Mesquite, acacia, locust, clover

• Prediction: Stomata mostly on bottom (hypostomatous)

Plant Type 3: Plant with simple broad leaves

• Examples: Mulberry, desert willow, sunflower, squash

• Prediction: Stomata mostly on bottom (hypostomatous)

Why test different types? Different plant groups evolved different strategies!

Step 2: Prepare Test Leaves

On each plant species, you'll prepare 3 leaves with different treatments:

Leaf 1 - Top Surface Blocked:

• Apply petroleum jelly ONLY to upper (top/adaxial) surface

• This blocks stomata on top (if present)

Leaf 2 - Bottom Surface Blocked:

• Apply petroleum jelly ONLY to lower (bottom/abaxial) surface

• This blocks stomata on bottom (if present)

Leaf 3 - Control:

• Don't apply anything

• Shows normal transpiration rate

Detailed application procedure:

1. Prepare petroleum jelly:

   • Warm slightly (easier to spread)

   • Have cotton swab or gloved finger ready

2. Apply to leaf surface:

   • Spread thin, complete coating

   • Cover entire surface edge to edge

   • Include major veins

   • Don't coat so thick it drips

3. Important tips:

   • Too thick: Adds weight (throws off measurements)

   • Too thin: Won't block all stomata

   • Just right: Thin, shiny, complete coverage

   • Avoid the petiole (don't block water transport)

4. Let dry 5 minutes before bagging

Labeling system:

• Grass - Top blocked

• Grass - Bottom blocked

• Grass - Control

• (Repeat for other species)

Step 3: Bag All Leaves

You'll bag 9 leaves total (3 per species).

Follow same bagging procedure as Experiment 1:

• Clear plastic bags

• Seal around petiole

• Label clearly with treatment

• Record start time

• Check for holes/gaps

Special considerations:

• Bag must not touch petroleum jelly coating

• May need larger bags if jelly makes leaf surface sticky

Step 4: Wait 4 Hours and Measure

Use the same measurement method as Experiment 1:

• Remove bags after exactly 4 hours

• Wipe water onto pre-weighed paper towels

• Calculate water collected (g)

• Calculate rate (g/hour)

Step 5: Record Your Data

Calculate % of Control:

• (Treatment rate ÷ Control rate) × 100 = % of Control

• Example: If control = 1.0 g/hr and bottom-blocked = 0.2 g/hr:

  • (0.2 ÷ 1.0) × 100 = 20% of control

  • This means bottom surface is responsible for 80% of water loss!

Analysis Questions:

For each species, determine where stomata are located:

1. Grass:

   • Top blocked reduced transpiration to _____ % of control

   • Bottom blocked reduced transpiration to _____ % of control

   • Conclusion: Stomata are on    Top only  /  Bottom only  /  Both surfaces

   • Pattern: _____ (hypostomatous/amphistomatous/epistomatous)

2. Compound leaf plant:

   • Top blocked reduced transpiration to _____ % of control

   • Bottom blocked reduced transpiration to _____ % of control

   • Conclusion: Stomata are on    Top only  /  Bottom only  /  Both surfaces

   • Pattern: _____ (hypostomatous/amphistomatous/epistomatous)

3. Simple leaf plant:

   • Top blocked reduced transpiration to _____ % of control

   • Bottom blocked reduced transpiration to _____ % of control

   • Conclusion: Stomata are on    Top only  /  Bottom only  /  Both surfaces

   • Pattern: _____ (hypostomatous/amphistomatous/epistomatous)

Synthesis:

4. Did different plant types show different stomata distributions?   Yes  /  No

5. Why might having stomata on the bottom be advantageous?

   • Bottom surface is _____ (cooler/warmer) than top

   • Bottom is more _____ (humid/dry) due to less air movement

   • Bottom receives less _____ (direct sunlight)

   • All these factors _____ (reduce/increase) water loss

6. Why might grasses have stomata on both surfaces?

   • Grasses need _____ (high/low) photosynthesis rates for rapid growth

   • More stomatal surface area = more _____ uptake

   • Trade-off: Higher water use, but _____

Arizona observation: Check your desert natives—many have extra adaptations like:

• Hairy leaf surfaces (traps humid air near stomata)

• Sunken stomata in pits (reduces air movement across pores)

• Thick cuticles (waxy waterproof coating)

Part 2: Analyze Hydraulic Architecture

Goal: Understand how plant "plumbing" varies with growth form. Tall plants, vines, and shrubs face different hydraulic challenges.

The physics: Water moves from roots to leaves through xylem vessels. Long pathways and narrow vessels create resistance. Plants must balance efficiency (moving enough water) with safety (preventing air bubbles/cavitation).

Step 1: Select 5 Different Plant Types

Choose plants representing different growth strategies:

1. Small herb or forb

• Examples: Small wildflowers, garden herbs (basil, marigold)

• Short pathway, few leaves

• Prediction: Low hydraulic demand

2. Grass

• Examples: Lawn grass, ornamental grasses

• Parallel venation, many narrow leaves

• Prediction: Moderate demand, efficient transport

3. Shrub or woody plant

• Examples: Brittlebush, creosote, sagebrush

• Multiple stems, moderate height

• Prediction: Moderate demand, woody vessels

4. Tall plant

• Examples: Tree, tall perennial (sunflower >6 ft)

• Very long pathway

• Prediction: High hydraulic challenge

5. Vine or climbing plant

• Examples: Grape, morning glory, cucumber

• Long, flexible pathway

• Prediction: Specialized vessels (wide for efficiency)

Arizona options:

• Herb: Desert marigold, globemallow

• Grass: Dropseed, blue grama

• Shrub: Brittlebush, creosote, bursage

• Tall: Palo verde, mesquite, century plant stalk

• Vine: Wild grape, gourd, melon

Step 2: Measure Each Plant

For each of your 5 plants, collect these data:

A. Plant Height

What to measure: Ground level to highest photosynthetic tissue (tallest leaf or green stem tip)

How to measure:

1. Find highest point of plant

2. Use measuring tape held vertically

3. Record in cm or inches

For tall plants:

• May need to estimate using reference objects

• Example: Stand at base, hold ruler at arm's length, use proportions

Recording:

• Plant height: _____ cm (_____ inches)

B. Count Leaves

What to count: All functional leaves (green, photosynthesizing)

For small plants (<50 leaves):

• Count directly

• Include all leaves from base to tip

For medium plants (50-200 leaves):

• Count one representative branch

• Count number of similar branches

• Multiply: (leaves per branch) × (number of branches)

For large plants (200+ leaves):

• Count leaves in 30 cm × 30 cm sample area

• Estimate total canopy area

• Extrapolate: (leaves per area) × (total canopy area)

Recording:

• Number of leaves: _____ (or estimated _____)

• Method used:  /  Direct count  /  Branch method  /  Area extrapolation

C. Estimate Total Leaf Area

What this measures: Total photosynthetic surface area—drives water demand.

Method 1: Photo analysis (most accurate)

1. Collect 3-5 representative leaves

2. Photograph each on white paper with scale

3. Use ImageJ to calculate area:

   • Tutorial: https://www.youtube.com/watch?v=kXFShlKSXbM

4. Calculate average leaf area

5. Multiply by total number of leaves

Calculation:

• Average leaf area: _____ cm²

• Number of leaves: _____

• Total leaf area = (average) × (number) = _____ cm²

Method 2: Geometric approximation (quicker)

For simple leaf shapes:

• Oval: Length × Width × 0.66

• Ellipse: Length × Width × 0.79

• Rectangle: Length × Width

• Triangle: (Length × Width) ÷ 2

Recording:

• Total estimated leaf area: _____ cm²

D. Measure Stem Diameter

What to measure: Main stem diameter at the thickest point near ground level.

Why this matters: Stem diameter indicates xylem capacity—wider stems can hold more water-conducting vessels.

How to measure:

1. Hold ruler next to stem

2. Measure diameter (width across)

3. Estimate to nearest mm

For irregular stems:

• Measure longest diameter

• Measure shortest diameter

• Average them

Recording:

• Stem diameter: _____ mm

Expected range:

• Herbs: 2-10 mm

• Grasses: 1-5 mm

• Shrubs: 10-50 mm

• Trees: 50-500+ mm

• Vines: 5-20 mm

E. Trace Water Pathway Length

What to measure: The actual distance water travels from roots to the highest leaf.

Why this matters: Longer pathways = more resistance = harder to move water against gravity and friction.

Detailed procedure:

1. Identify the highest leaf:

   • This is the farthest point water must reach

   • Mark it with tape if helpful

2. Start at ground level:

   • This is where roots meet the stem (root collar)

3. Trace the main pathway:

   • Use flexible measuring tape or string

   • Follow the main stem upward

   • When stem branches, follow the branch leading to highest leaf

   • Continue to the target leaf

4. Measure total distance:

   • If using string: Straighten it and measure with ruler

   • If using tape: Read measurement directly

   • Record in cm

Tips:

• Follow the ACTUAL path (curves, zigzags)

• Don't measure straight-line distance (that's not how water travels!)

• For vines: Follow the twisting pathway

Recording:

• Water pathway length: _____ cm

Expected values:

• Small herbs: 5-30 cm

• Grasses: 10-50 cm

• Shrubs: 50-200 cm

• Tall plants: 200-500+ cm

• Vines: Highly variable (50-1000+ cm)

F. Calculate Path:Area Ratio

What this means: Pathway length per unit leaf area—indicates hydraulic efficiency.

Formula: Path:Area Ratio = (Pathway length in cm) ÷ (Total leaf area in cm²)

Example calculation:

• Pathway: 150 cm

• Leaf area: 500 cm²

• Ratio: 150 ÷ 500 = 0.3 cm/cm²

Interpretation:

• Low ratio (<0.5): Efficient—short pathway relative to leaf area

  • Example: Short, leafy herb

• High ratio (>2.0): Challenging—long pathway relative to leaf area

  • Example: Tall tree or long vine

Recording:

• Path:Area ratio: _____ cm/cm²

• Interpretation:  /  Efficient  /  Moderate  /  Challenging

G. Examine Stem Xylem Pattern

What to look for: The arrangement of water-conducting vessels in the stem.

Note: This requires cutting the plant. Only do this if:

·         You have permission

·         Plant is common/not endangered

·         You're removing a small, expendable twig

If you can safely collect a small stem sample:

1. Cut a small twig or branch:

   • Use clean, sharp pruning shears

   • Cut at 45° angle

   • Choose branch about pencil-thickness
     
     
     
     
     
     
     

2. Make a fresh cross-section:

   • Cut straight across (perpendicular to length)

   • Make cut as smooth as possible

   • Wet the surface (helps see details)

3. Examine with hand lens:

   • Look for circular holes (vessels)

   • Note size and distribution

4. Identify vessel pattern:

Ring-porous:

• Large vessels in a ring around the edge

• Smaller vessels toward center

• Common in: Oaks, ashes, desert hardwoods

• Function: Large vessels in spring (high flow), smaller vessels for safety

Diffuse-porous:

• Vessels of similar size scattered throughout

• No obvious ring pattern

• Common in: Maples, most shrubs, creosote

• Function: More consistent flow year-round

Radial/Sectored:

• Vessels arranged in radial lines from center

• Common in: Some desert shrubs

• Function: Compartmentalization (if one sector fails, others continue)

Take a photo.

Recording:

• Vessel pattern:  /  Ring-porous  /  Diffuse-porous  /  Radial  /  Uniform  /  Could not determine

• Notes: _____

Arizona observation: Many desert plants have very narrow vessels (safer against cavitation in drought) or grouped vessels (if some fail, others compensate).

Step 3: Organize Your Data

Create a comprehensive comparison table:

Part 3: Observe Water Stress Responses

Find plants that are wilting from lack of water, document their stress responses, and watch them recover after watering.

Step 1: Find Water-Stressed Plants

Look for plants showing these signs:

• Wilted, drooping leaves

• Leaves folded or rolled up

• Leaves hanging down instead of spreading out

• Dull or greyish color instead of bright green

Find at least 3 different stressed plants (more is better).

Step 2: Document the Stress

For each stressed plant:

A. Take Photos

• Take clear photos from multiple angles

• Include something for scale (ruler or coin)

• Take close-ups of wilted leaves

B. Measure Leaf Angle

• Use a protractor or estimate

• Measure angle from horizontal:

  • 0° = leaf perfectly horizontal

  • 90° = leaf hanging straight down

• Measure 3-5 leaves and average

C. Describe Leaf Changes

Check all that apply:

☐ Leaves drooping/hanging

☐ Leaves folded along midrib

☐ Leaf edges rolled inward

☐ Leaves twisted

☐ Color change (dull, grey, brown)

☐ Other (describe): _____

D. Test Soil Moisture

Squeeze test method:

1. Dig down about 2 inches (5 cm)

2. Grab a handful of soil

3. Squeeze firmly

4. Classify:

   • Dry: Soil powdery, won't hold shape

   • Moist: Soil holds shape but crumbles easily

   • Wet: Soil holds shape, feels damp

   • Saturated: Water drips out when squeezed

E. Find Comparison Plant (Control)

Find a healthy, non-stressed plant of the same species:

• Measure its leaf angle

• Note its appearance

• Check its soil moisture

Step 4: Record Your Observations