Vacuoles - Development, Types, Functions and Microscopy
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Vacuoles

Development, Types, Functions and Microscopy


What is a Vacuole?


A vacuole is a membrane bound, multifunctional organelle found in the cells of plants (including algae and fungi) and some protists and bacteria. Vacuoles are acidic in nature and share some basic properties with lysosomes that are predominantly found in plant cells. Depending on the type of plant, there are different types of vacuoles with specific properties that are crucial to their functions.

* Unlike lysosomes in animals, there is only one of a few vacuoles in individual plant cells.

* Vacuoles take up 80-90 percent of the entire plant cell volume


Development (Biogenesis of Vacuoles)



Vacuoles are complex organelles and much about their biogenesis remains unknown. However, studies suggest that vacuoles found in the root tips originate from vesicles that arise from Golgi bodies.

Here, the process involves the fusion of these vesicles to produce prevacuoles, which are vacuole precursors. It is the fusion of these prevacuoles that ultimately results in the formation of a vacuole. 


Types and Functions of Vacuoles



Lytic vacuoles

Lytic vacuoles share similar properties with lysosomes found in animals. As such, they contain different types of hydrolytic enzymes responsible for the degradation of such molecules as nucleic acids, proteins and polysaccharides. Researchers have suggested that these particular organelles either originate from the trans-Golgi network or the dilation of a part of the smooth endoplasmic reticulum.

These types of vacuoles are also referred to as lytic compartments and are characterized by the optimum pH of 5. Research studies have found lytic vacuoles to contain the following types of hydrolytic and oxidizing enzymes:

  • Hydrolases - Essentially, hydrolases are different types of hydrolytic enzymes that use water to break up chemical bonds. This allows them to divide larger molecules into smaller ones.
  • Esterases - This includes hydrolase enzymes that specifically serve to break down esters (compounds made up of an acid and alkyl group) in to acids and alcohol group.
  • Nucleases - Enzymes responsible for breaking down the bonds (phosphodiester bonds) to produce nucleotides.
  • Peroxidases - Peroxidases include enzymes that typically break down hydrogen peroxide, removing it from chloroplast and cytosol among others in plants.

There are different processes through which cells eliminate unwanted/old material, unwanted cytoplasm or the entire cell.

In plant cells, this includes:

Autophagy

In plants, autophagy is an important process that helps in the elimination of unwanted material from the cells. Here, various materials in the cytoplasm that are no longer required by the cell are enclosed within a vesicle refered to as the autophagosome and transported to the vacuole where they are degraded.

The invagination of the double membrane of the autophagosome makes it possible for this vesicle to enclose and hold cytoplasmic material/components to be delivered to the vacuole. This process is also involved in recycling of material.

By breaking down various cell components, they are reduced to their basic components that can then be used by the cell. For instance, the breakdown of proteins produces peptides that can later be transported through the endoplasmic reticulum and Golgi apparatus to process proteins.

Autophagy in cells occurs in response to different conditions within the cells or in response to factors affecting the body in general. For instance, such stressful conditions as starvation result in the degradation of various components of the cell such a proteins and even lipids in order to produce energy.

While it was previously believed that autophagy non-selectively eliminates various components in the cell, recent studies have shown that this mechanism can and does selectively eliminate given components such as proteins in specific conditions or in response to given stressful conditions in yeast cells.



Cell Defence and Cell Death


Vacuoles play a crucial role in the defense and death of cells.

Although the process is yet to be fully understood, vacuoles play an important role in immunity of the cell by releasing various enzymes (hydrolytic enzymes) and antimicrobes that destroy the invading pathogen. However, the mechanism has also been associated with programmed cell death (PCD).

In reaction to invading organisms in the cell, an enzyme refered to as vacuolar processing enzyme triggers the disruption of the vacuolar membrane, causing the vacuole to collapse and release hydrolytic enzymes and other antimicrobes. This not only results in the destruction of the invaders, but also the cell itself.

On the other hand, the fusion of the central vacuole with the plasma membrane in the presence of proteasome can cause the vacuole to release antibacterial protease as well as other vacuole components that can cause the death of the cell.


Protein storage vacuoles (PSV)

Protein storage vacuoles can be found in the storage tissues where they accumulate proteins. Seeds are good examples of tissues where reserve proteins are stored. All proteins to be stored are first synthesized in the rough endoplasmic reticulum and then transported to the protein storage vacuole (PSV).

In some plants, this process involves the transport of proteins through autophagy and protein bodies (PBs). On the other hand, they may be released from the Golgi apparatus (having been synthesized in the ER) as prevacuoles before arriving at the vacuole for storage.

For proteins to be successfully transported from the Golgi apparatus to the vacuole, protein targeting is essential. Here, peptide targeting sequence target given receptors on the vacuole, which allows for proteins to be successfully transported and stored.

Depending on the type of plant, storage tissues (seeds, etc) will contain many, densely packed protein storage vacuoles. Moreover, depending on the plant, there may be one or different types of proteins (sub-domains) stored.

Gas vacuoles

Gas vacuoles are composed of hollow cylindrical gas vesicles. They are typically found in bacteria and have a permeable membrane that allows air to pass through. This membrane also serves to bind the vesicles. These vesicles can either inflate (fill with air) or deflate allowing the bacteria such as cyanobacteria to float or remain at a given desired depth in water. 


Contractile vacuoles

Contractile vacuoles are membrane bound organelles that are typically found among members of kingdom protista (algae, amoebas, and ciliates etc). In these cells, the contractile vacuole is particularly important given that it helps in osmoregulation (regulation of osmotic pressure).

Although the entre mechanism is yet to be understood, researchers suggest that the contractile vacuole system (contractive vacuole complex) functions through the activities of two compartments that are bound by two differentiated membranes.

The two membranes have different properties that make it possible for the vacuole to carry out osmoregulation. Here, the first membrane is divided into many vesicles and tubules and contains numerous proton-translocating V-ATPase enzymes. These components are responsible for producing an electrochemical gradient of protons and fuse with the membrane of the second compartment.

The second compartment expands into the reservoir for fluid storage and can fuse with the cell membrane of the cell. However, it lacks the V-ATPase holoenzymes. As such, it undergoes contraction periodically which enables the vacuole to expel fluids. Together with other solutes, this system works like a pump that pumps out excess water from time to time to prevent the cell from swelling and getting ruptured.   



Sap vacuoles

The sap vacuole is also commonly refered to as the central vacuole of a cell. It is the large, central organelles that occupy most part of the cell volume. This organelle contains the fluid known as the cell sap, which consists of such contents as water, sugars, minerals and amino acids among others. As a plant and cells mature, provacules from the Golgi complex fuse to form the sap vacuole at the center of the cell.

Contents of the cell sap are transported to the vacuole from the cytoplasm in the cell.

Some of the other functions include:

  • Cell growth - Vacuoles are important in plant cells given that they help maintain turgidity of the cell. Enlargement of the vacuole consequently results in growth/increased size of the cell. This ultimately contributes to tissue rigidity.
  • Storage - Apart from proteins, vacuoles are storage compartments for metabolites, organic acids and sugars among others
  • Pigment deposition - Vacuoles are a site where pigments are deposited allowing for such vegetable colors as red, blue, scarlet etc.


Microscopy


A light microscope can be used to view and study the structure of a vacuole. Although the vacuole does not stain as other organelles of the cell (because the vacuole does not contain much stainable constituents) experiments have shown that it is possible to stain this organelle as the sap inside the vacuole take in and accumulate colored dyes. Most of the dyes used are basic in nature. However, the pH level of the dye also depends on the type of the vacuole being stained.

Some of the dyes that have been used for the purposes of staining vacuoles include:

  • Neutral red
  • Brilliant cresyl blue
  • Methylene blue
  • Pyronin

See more on Cell Staining.

Here, we will focus on staining using Neutral red solution (pH less than 6.0).

Requirements

  • A part of a plant (roots, leaves etc)
  • Neutral red stain (7.2 in 0.08M phosphate buffer)
  • Water (distilled)
  • A compound light microscope
  • Glass slides and cover slips
  • Droppers
  • Tweezers


Procedure

Obtain a thin skin of the plant using a pair of forceps and stain the sample with 0.01 percent of neutral red

Rinse the sample with the phosphate buffer

Mount for observation

Observation

Through this procedure, the stain will only stain the vacuole of live plant cells without staining any other organelle. When viewed under the microscope, students will view the vacuoles appear deep red in color.

Take a look at other biological parts of the cell like:  Ribosomes, Mitochondria, Lysosomes, Golgi Apparatus

Return from Vacuoles to Cell Biology





Sources

Deepesh N. De (2000) Plant cell vacuoles.

Misoon Park, Soo Jin Kim, Alessandro Vitale, and Inhwan Hwang (2004) Identification of the Protein Storage Vacuole and Protein Targeting to the Vacuole in Leaf Cells of Three Plant Species.


Links

https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/vacuole

http://www.cell.com/current-biology/abstract/S0960-9822(14)01550-4


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