BIO 43 - PLANT PHYSIOLOGY

Exercise V. Stomatal Regulation

SUBIMTTED TO: Mr. PERCY MARK SIENES

Laboratory Instructor

SUBMITTED BY: JAIKA DE JESUS YBER

BS Biology IV
Abstract

In this experiment, we focus on the mechanism that helps regulate gas exchange and water

regulation. Leaf samples from Bangka-bangkaan plants were used to see the epidermal layers showing the

opening and closing gates of the guard cells in the stomata in dark conditions and light exposure.

Introduction

Just like any living organisms response to keep homeostasis, they have specialized cells to

perform certain tasks. Two specialized cells called ‘guard cells’ make up each stoma. Plants have many

stomata (up to400 per mm2) on their leaf surfaces and they are usually on the lower surface to minimize

water loss. In humans and animals, they have pores to execute these processes. As for plants, they have

stomatas. The stomata are functional units of the epidermis with the primary control mechanisms that

plants use to reduce water loss and gas exchage between intercellular spaces. Stomata are sensitive to the

environmental cues that trigger the stomata to open or close. It is well-known that stomata open in a

humid surrounding, and close when it is dry. The stomata of most plant species are also closed in

darkness while light stimulates opening.

Throughout this whole study, we just focus on the stomata of terrestrial plants. The model

organism used for this stomatal regulation experiment is the Rhoeo spathacea commonly known as

Bangka-bangkaan. Using its leaves, the epidermal layers can be observed where you can find the stomata

and their specialized guard cells. The guard cells are those that regulate the stomatal pore activity by its

shape. If the guard cells are flaccid, the stomata is closed. But if the guard cells are turgid, the stomata is

seen as open (Every & Eichhorn, 2013). The composition of water in the guard cells is what makes them

change the shape of the stomata making them act similar to hydraulic valves (Taiz & Zeiger, 2005).
 Stomata of Rhoeo spathacea were observed in the different conditions of the presence of

or absence of light. Other factors that might affect the stomata could be temperature or humidity.

Methods

Distilled water was poured into two petri dishes, just enough to cover the surface of the each.

Using a ruler to mark measurements and a pair of scissors to cut, the leaves of Rhoeo spathacea were cut

approximately 2-3cm in width. Only twelve pieces was used for this experiment. Carefully scraped off the

upper epidermis, palisade layer, and spongy layer with a scalpel, the lower epidermis can be seen under a

microscope where the stomata can be found. The thin layer of the leaf will allow easy staining.

For the dark conditioned samples, 6 pieces were placed into one of the petri dishes. It was then

breathed on, covered by aluminum foil, and then placed at a dark area inside a cabinet.

A 5cm petri dish filled with 2ml of 2N KOH was placed into the center of a 9cm petri dish

slightly filled with distilled water. The rest of the 6 pieces were placed onto the distilled water with its

lower part facing upwards. On top of the 9cm petri dish, ice was placed and left on top of the apparatuses

with the maize samples for approximately 30 minutes. The other sample was then exposed to light with

the use of a 100-watt bulb for 30 minutes.

While waiting, ice cold 20 mM CaCl2, ice cold cobalnitrite stain, distilled water, and 5%

ammonium sulphite were prepared for staining. The leaf sections were then submerged in the ice cold 20

mM CaCl2 solution for 2 minutes. Afterwards, the sections were rinsed with distilled water and then

submerged again in the cobalnitrite stain for 3 minutes. The sections were again rinsed in distilled water

after this staining and then examined under the microscope. The areas that had black precipitate were

examined for stomata, as the black precipitate is the result of the reaction of potassium reacting to the

stains.
Results

According to classmates’ results;

Plant Stomatal activity

Rhoeo spathacea laves Light treatment Dark treatment

3
4

Dark Set-up No. Of closed stomata (HPO)

Sample 1 4/4

Sample 2 none

Sample 3 2/3

Sample 4 3/3
 Sample 5 2/3

Sample 6 3/4

Light Set-up No. Of opened stomata (HPO)

Sample 1 4/5

Sample 2 3/3

Sample 3 4/4

Sample 4 4/5

Sample 5 1/2

Sample 6 3/3

Conclusion

Through this experiment, we understood the role of potassium ions in affecting the water

potential levels and turgidity of the guard cells found in the stomata. Stomata are affected by

environmental cues that trigger it to open or close. Increased turgidity in guard cells causes stomatal

opening when exposed to light. The samples in the dark conditions had shown closed stomata. Light is

indeed important for plants to signal the guard cells to open the stomata for gas exchange and carbon

dioxide absorption for photosynthesis.