Title: Plant Aquaporins: How Water Moves Into and Out of Plant Cells Authors: Janese Jones and Hope Diamond Course: BI589, Spring 2003 Instructor: Dr. Lucinda Swatzell Abstract: The classic model for water movement across plant cell membranes is

based on osmosis. However, diffusion rates over biological membranes do not equal

the rate of water flow into and out of plant cells. Instead, a new model has been

proposed that is based on a newly identified family of membrane proteins, aquaporins,

which specifically channel water across cell membranes. The specialized structure of

these proteins facilitates rapid water movement. Plants can control water movement by

controlling aquaporin function and distribution.

AQUAPORINS IN PLANTS

An Outdated Model: Osmosis Across Membranes

All living organisms are composed of one or more cells. These cells have a ‘skin’

called the plasma membrane that protects them from the outside environment. The

plasma membrane regulates the movement of water, nutrients and wastes into and out

of the cell. It is comprised of lipids and proteins (Fig. 1A). Because lipids are not

miscible in water, the plasma membrane is an effective barrier to mass flow. Therefore,

salts, sugar, proteins, and many other compounds move across the membrane through

protein channels and transporters (Fig. 1B). However, these proteins channels and

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transporters are selective. Thus, the plasma membrane is partially permeable so that

some substances cross more easily than others (Lackie and Dow, 1999).

Figure 1A. The plasma membrane is comprised of a bilayer of phospholipids. Phospholipids have an alcohol, hydrophilic (water-loving) head (balls) and fatty, hydrophobic (water-fearing) tails (black lines). The hydrophobic tails face each other and form a water resistant barrier.

Figure 1B. Ions, sugars, and proteins must move through or across proteins channels and transporters in the plasma membrane. Many types of compounds are excluded by the lipid bilayer. This makes the plasma membrane selectively permeable to compounds in its environment.

Water is the major constituent of all living cells and of the environment that

surrounds them. Water transport across cell membranes is therefore crucial for

biological function, especially for those physiological processes that involve fluid

transport. The classic model for cell water balance is based on osmosis (Fig. 2).

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Figure 2. Osmosis is the diffusion of water along a concentration gradient. Water flows from areas where water is more concentrated to areas where water is less concentrated. In plant and animal cells, water may move across the lipid layer, but does so slowly and cannot account for the actual rate of water flow into or out of cells.

Osmosis is the diffusion or net flow of water from a region of high water concentration to

one of low water concentration (Lackie and Dow, 1999). Although water diffuses

through lipid bilayers, the water diffusion rate does not match the demand for water for

many physiological processes and does not equal the actual rate of water transport into

live cells (Philip, 1958). Instead, a family of membrane channel proteins facilitates rapid

transport of water across biological membranes. These proteins, termed “aquaporins,”

are found in all forms of life: the archaeabacteria, eubacteria, fungi, plants, and animals.

A Very Old Protein, An Important New Discovery

The ubiquitous aquaporin family of transmembrane channel proteins dates back

perhaps 2.5 to 3 billion years in evolutionary time. It arose by an intragenic duplication

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event that preceded diversification of the family (Park and Saier, 1996). Still, its

existence was not documented until recently. In 1958, Philip determined that diffusion

rates across plasma membranes in oat coleoptiles did not account for water flow rates.

This challenged the idea that water flowed directly across biological membranes.

However, no alternative hypothesis was developed. Finally, in 1984, water channel

proteins were discovered in by two separate researchers. Gorin et al. (1984) revealed

that protein channels in cow lens fibers allowed rapid water movement into cells.

Simultaneously, researchers studying water balance in erythrocytes (Macey, 1984)

proposed that an analogous protein existed in red blood cells. Agre et al. (1987)

isolated a 28 kDa protein during the isolation and characterization of the Rh factor in red

blood cells. The 28 kDa protein shared structural and functional features of the protein

in cow lens. Later, the water conductance properties of both new proteins were

demonstrated through expression of the protein in frog oocytes (eggs). Expression in

frog eggs resulted in mass inward water flux and caused rupture (Preston et al., 1992).

The water transport capabilities of these newly discovered proteins were self-evident.

The possible existence of aqueous pores in plant membranes was discussed in the

early 1960’s by Ray and Dainty (Maurel, 1997). The first plant aquaporin (Chrispeels

and Agre, 1994), named tonoplast intrinsic protein (TIP) was identified because of its

abundance in the membranes that surround the protein-storage vacuoles of the storage

parenchyma cells of bean seeds.

Structure and Function

Aquaporins are specific for water transport. They are small proteins

(between 26 and 30 kDa) that typically contain six membrane-spanning α-helices, with

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the N-and C-termini both located on the cytoplasmic side of the membrane (Fotiadis et

al., 2001; Fig. 3A). The helices form a channel through which typically only water can

flow. In addition, aquaporins contain "on/off" switches that are controlled by the addition

or removal phosphate ions (Maurel et al., 1995; Johansson et al. 1998). Structurally,

the two halves of the polypeptide show obverse symmetry, with the loops containing the

NPA (amino acids: asparagiNe-Proline-Alanine) motif overlapping in the middle of the

lipid bilayer (in the so-called “hourglass” model) to form two hemipores that together

create a narrow, water-filled channel. This distinctive two-fold axis of symmetry

accounts for the fact that aquaporins can mediate bi-directional water flow, in contrast to

many ion channels (Tyerman et al., 1999). The channel itself is sensitive to mercury,

and the entire function of the molecule can be inhibited by exposure to mercury. With

exposure to mercury, the shape of the central channel is changed, and water flow is

occluded (Maggio and Joly, 1995; Heymann et al., 1998). Because of their structure,

aquaporins transport water 10 to 20 times faster than diffusion across membranes (Fig.

3B). The most active aquaporins are highly specific for water and may transport up to 1

billion water molecules per second per 28-kDa protein subunit, depending on the

osmotic gradient imposed. Some members of the MIP family transport glycerol as well

as water, whereas other members of the family found in bacteria and yeast transport

only glycerol and neutral solutes (Maurel, 1997).

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The Plant Aquaporin Family

Aquaporins belong to one family of proteins, the major intrinsic protein (MIP)

family. There are three major divisions of aquaporins based on their distinct location.

The first major division is the plasma membrane intrinsic proteins (PIP). The PIP’s are

proteins that can be found in the plasma membranes of plants and animals. The

second major division of aquaporins is the tonoplast intrinsic protein (TIP). The TIP’s

are known to be components of the vacuolar membrane (tonoplast) (Schaeener, 1998).

The tonoplast is a membrane in plant cells that surrounds the vacuole. The tonoplast is

selectively permeable and regulates the concentration of ions in the cell cytoplasm. The

Figure 3A. Aquaporins are comprised of six membrane spanning helices that form a channel through the membrane. The center of the channel is formed by the overlap of two essential opposing portions of the protein that allow bidirectional water flow. The nature of this center region also excludes other molecules, including ions.

Figure 3B. As a general rule, the rate of movement of water through aquaporins is 10-20 times faster than osmosis over a membrane, but true rates are species specific and vary with cell function.

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TIP was the first aquaporin identified in plants. The third major division of aquaporins is

the Nod26-like intrinsic protein (Chrispeels and Maurel, 1994). The Nod26 is found in

the symbiosome membrane surrounding nitrogen-fixing bacteria (Miao and Verma,

1993; Chaumont et al., 2000).

Plant cells have aquaporins in both their plasma and vacuolar (tonoplast)

membrane. The abundance of aquaporins in these membranes is undoubtedly

regulated at the transcriptional level (Kaldenhoff et al. 1993, 1995). As in animals, plant

aquaporins exhibit highly specialized developmental patterns (Opperman et al., 1994;

Mundree et al., 1996). For example, the α-TIP is specifically expressed in seeds and is

also present in the membranes of protein storage vacuoles during the late and the early

stages of seed maturation and germination (Maurel et al., 1995).

Plants, Water Movement, and Aquaporins

Plant-water relations and water flow in plant tissues have been well-

characterized (Slayter, 1967), but the presence of water pores or even proteinaceous

water channels in plant membranes was not established until fairly recently (Steudle,

1992). Water is taken up from the soil through the roots, where it flows from the cortex

into vascular tissues. Water can move through the cortex between root cells or through

root cells, but all water must cross cell membranes to enter the plant vascular system

(Fig. 4).

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Figure 4. Root cross section. Water may enter the root through root hairs (1), between root cortex cells (2), through root cortex cells (3), or through symbiotic fungal hyphae (4). Irrespective of the pathway, all water must pass over the endodermis (central cylinder; gray cells). Water must enter the endodermis cells via aquaporins.

The plant vascular system is comprised of many tubular cells arranged end to

end so that long conducting tubes are formed. The water conducting tissue in the

vascular system is called xylem and the sap conducting tissue is called phloem. The

tubular xylem full of water is a continuous column that extends from the roots to the

leaves. In the leaves, it extends through the leaf mesophyll to the stomata, gas

exchange pores, and is lost to the atmosphere by evaporation. This evaporative pull

and the cohesiveness of water generate the tension that pulls the water upward through

the plant (Fig. 5).

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Figure 5. Water molecules cohere to each other and adhere to the vascular tissue cells. This forms a continuous column of water. Water evaporates from leaves when stomata, gas exchange pores, are open. This places tension on the water column and water is pulled from the root cortex and subsequently out of the soil. Water uptake is enhanced through aquaporin control and expression in vascular tissue.

The presence of aquaporins in cellular membranes may facilitate a transcellular

pathway for water flow. Plants can control the flow in several ways. Aquaporin

function can be regulated by phosphorylation, but the easiest way for plants to control

aquaporins is through quantity (gene expression) and distribution. The more

aquaporins present in a given tissue at a given time, the more water flow is facilitated.

Conclusion

Within the next century, water quality and availability concerns will supercede

concerns for other resources, including energy. As desertification continues and/or a

potential ice age threatens, and the human population grows exponentially, water

resources will become even more limiting. Current research on aquaporins in plants

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focuses on the role of aquaporins in abiotic stress conditions (high salinity, high

temperature, aridity, and cold). Because water is a major component of all living cells,

including plants, our oxygen and food supply, the future study of aquaporins in plants is

our future too.

Literature Cited

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