Protocol

Author

Rana Munns

Overview

This protocol describes how to measure and record the water content of plant organs, on a dry weight basis, and the relative water content (RWC) of organs relative to their fully hydrated status, also on a dry weight basis.

Background

Water content (WC)

Water content is a slight misnomer, it is really a water ratio. It is the amount of water in the leaf relative to its dry weight.

Water content should not be expressed on a fresh weight basis. The ratio of H₂O/(H₂O+DW) is a mathematical and biological absurdity for most species as the leaf H₂O is much greater than the DW. A small change in water content per leaf can have no statistically significant effect when expressed on a fresh weight basis, but a significant effect when expressed on a dry weight basis.

When water content is expressed relative to unit leaf area, it is called succulence

 

Relative water content (RWC)

RWC is the water content of a given amount of leaf relative to its fully hydrated or fully turgid state. It indicates the hydration state of the leaf.

RWC has two advantages over WC.

1. It is independent of anatomy, as it is the uptake of water related to the DW of that particular segment. For example, RWC does not vary along a leaf, although the WC does.
2. Changes in RWC are proportional to changes in turgor of a leaf, and so becomes an indirect measure of the change in turgor under certain conditions such as a constant elastic modulus (E). As the cell swells and shrinks due to water uptake or loss, the turgor change is determined by the elasticity of the cell wall.

Historically, the water content of tissues was expressed relative to their fully turgid water content when floated on water (Weatherley, 1950; Barrs and Weatherley, 1962). This was first termed ‘relative turgidity’ and later called ‘relative water content’ (RWC). Weatherley (1950) had suggested the method because it was simple and did not require specialized equipment. He tacitly assumed that the cell solute content was constant, but the later discovery of osmotic adjustment made this assumption less tenable (Boyer et al., 2008). Osmotically adjusted cells can have the same turgor and thus hydration as their non-acclimated counterparts. When the water content was measured by floating excised tissue on water to attain the fully hydrated condition (Weatherley, 1950), theory indicates that the turgor of osmotically adjusted cells would rise higher than normal. Likewise, water contents would become higher than normal. Hence this method should not be applied to tissues that are likely to have osmotically adjusted. This particularly applies to plants adapted to saline soils where Na⁺ and Cl^– accumulation can allow osmotic adjustment (Boyer et al., 2008).

Materials/Equipment

For measuring WC:

• electronic balance
• capped and weighed vials
• razor blade
• drying oven (70^oC)

For measuring RWC:

• all of the above plus Petri dishes
• dark humid chamber, or mannitol of the same water potential as in the soil

Units, terms, definitions

Water content – WC

Relative water content – RWC

Turgid weight – TW

Dry weight – DW

Fresh weight – FW

Procedure

Method of measuring WC:

Fresh weight:

• Weigh the capped vials; number both vials and caps. These can be 22 ml glass specimen tubes or 10 ml or 15 ml plastic autoclavable tubes. All vials must be weighed as they vary in weight significantly.
• Take tared (pre-weighed) capped vials to plant, and detach a segment of leaf (monocot) or whole leaf (dicot) or root after harvesting and blot dry. Cap immediately after organ is placed in vial. Store on ice if unable to return to electronic balance quickly.
• Weigh capped vial with plant tissue.

Dry weight:

• Take off cap. Dry leaf in vial overnight at 70^oC.
• After 24 h, or constant weight, replace cap as soon as removed from oven, let cool to room temperature, then weigh.

Calculate:

(FW-DW)/DW (see below table.)

Method of measuring RWC:

Fresh weight: measure as above

Turgid weight:

• Rehydrate freshly weighed leaves for 3-4 hours, by either floating in a Petri dish in water for about 3 h, or standing in a humid atmosphere, or until weight gain is complete.
• Replace in capped vial and weigh.

Dry weight: measure as above

Sample calculation:

A	B	C	D	E	F	G	H
Sample	Vial no	Vial weight (g)	Weight of freshly cut leaf + vial (g)	Weight of rehydrated leaf + vial (g)	Weight of dried leaf + vial (g)	WC (g/g) (D-C)/F-C)	RWC (g/g x 100) (D-F)/(E-F)
Replicate 1	1	10.9343	11.1440	11.1791	10.9745	5.21	82.8
Replicate 2	2	11.0317	11.1769	11.2001	11.0575	5.63	82.8

During the rehydration process, observe closely, using a dissecting microscope, and if air spaces appear infiltrated (which may occur due to excess swelling and leakage if leaves have undergone large osmotic adjustment), use alternative methods such as:

• Rehydrate whole plants by placing pot in dark humid chamber for one hour. Do not leave longer as osmotic potential will change due to respiration consuming the sugars.
• Float detached leaves in mannitol of the same water potential as in the soil.

Other resources

Dark chamber to place pots to allow rehydration of intact plants

Notes and troubleshooting tips

Do not wait more than 4 h for rehydration, as respiration and carbohydrate metabolism can reduce the concentration of soluble sugars and therefore of osmotic pressure.

If measuring the RWC of growing tissues, carry out the rehydration step at 4 degrees C, or an appropriate temperature to inhibit growth.

A herbaceous dicot leaf has a water content of 10-12 g H₂O per g DW, and a monocot leaf of 6-7. The WC of the leaf of a woody perennial is much lower, 3-4 g H₂O per g DW. With stress, these values decrease by a third.

The WC varies along a leaf, being greater at the base and lower at the tip. This is not just due to effects of transpiration, it reflects the different anatomy of cells and tissues along the leaf. Either a whole leaf should be sampled, of a known age or stage of development, or a defined portion of the leaf should be taken, eg a segment in the middle.

Unstressed leaves will have a RWC of 90-95 % depending on the humidity (vpd) and light. Stressed and wilted leaves may drop as low as 50 %. Few leaves can recover from a RWC of 40 %.In the dark, the RWC will be about 99 %.

Literature references

Barrs HD, Weatherley PE. 1962. A re-examination of the relative turgidity technique for estimating water deficits in leaves. Australian Journal of Biological Sciences 15, 413-458.

Boyer JS, James RA, Munns R, Condon AG, Passioura JB. 2008. Osmotic adjustment may lead to anomalously low estimates of relative water content in wheat and barley. Functional Plant Biology 35, 1172-1182. doi: 10.1071/FP08157

Weatherley PE. 1950. Studies in the water relations of the cotton plant. I. The field measurement of water deficits in leaves. New Phytologist 49, 81-97.

Health, safety & hazardous waste disposal considerations

Take care when handling razor blades