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Plant Detectives Manual: a research-led approach for teaching plant science

Activity 9: Drought response

9.1) Introduction and objectives

The previous activities have compared the structure and function of plants under normal growth conditions. Sometimes the effects of mutations are not apparent unless a plant is challenged in some way. By following the protocol below, you will analyse the response of both wild type and mutant plants to drought stress. In addition to following the growth and development of you plants during and after the drought, you will determine the relative water content of your plants.

The major goals of Activity 9 are to:

  1. investigate the effect of water deficit on wild type plants
  2. to compare these to the effects on the mutant.

You could also perform some of the assays from the previous activities if you think they can provide useful information. Discuss your interests with your peer mentor and instructor.

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9.2) Materials

  1. plants in pots: in general, plants between four to five weeks old are optimal for the drought experiment. The growth rate may vary, however, among batches of plants from different years and in different growing conditions. Teaching staff will advise you as to which of your plants (first or second round of sowing) are best for this assay
  2. balance (one–two per class)
  3. aluminium foil
  4. plastic Petri dishes (20)
  5. permanent marker (one)
  6. oven 60 ºC to 80 ºC
  7. Whatman filter paper (few pieces)
  8. scissors

9.3) Procedure

9.3.1) Experiment set up

Materials

Method

Trays, plants

  1. Move at least six plants of each genotype into two separate trays. Randomly place a combination of the wild type and mutant plants in each tray (i.e., don’t have half on one side of the tray and half on the other). Remaining plants can be used for other assays.

Labels, marker

  1. Label one tray as ‘WS–Drought; do not water’. Label the other tray as well-watered ‘WW–Control’.

 

  1. The drought treatment will be initiated for you seven days before the practical, when teaching staff will water the trays with 200 millilitres (ml) of water instead of 800 ml. You will start your observations on day seven of the drought, but you will be able to check your plants during lunch on each day in the following week. Depending on the performance of the plants, you may need to continue observations and measurements until the end of the course.

 

  1. Observe the growth and development of wild type and mutant plants after the drought treatment is imposed. Note any differences in phenotype between these two genotypes during water-deficit conditions.

9.3.2) Relative water content determination

When measuring plant water content, raw water content is not useful for comparison between plants with different morphologies. Instead, this information can be normalised by determining the relative water content (RWC) as:

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Where Fw is the fresh weight at harvest, Dw is the dry weight of the sample, and Tw is the turgid weight, or the maximum weight at the highest level of water holding capacity. On one day of the drought treatment (to be decided along with your supervisor), you will perform the determination of RWC as follows to check for differences in:

  1. the effect of the growth condition on the water content of the wild type plant
  2. the effect of the genotypes on the water content at both conditions.

 

Materials

Method

Scissors, plants

  1. Detach the rosette from the roots, carefully removing traces of soil. Aim to use at least five individuals of each genotype under both control and drought conditions (total 20 plants). Make sure plants are labelled clearly to help organise the data organised. Consider design principles when selecting plants.

Scale

  1. Record the Fw at time 0.

Plastic Petri dishes, H2O

  1. Place the rosettes in a Petri dish half-filled with water. Leave the rosette in the dish (incubate) for at least four hours at 4 ºC in the dark. You can also perform this incubation overnight and weigh the samples the next day.

Blot paper

  1. Blot the leaves and record the Tw.

80 ºC oven

  1. Place the rosettes in paper envelopes and dry at 80 ºC overnight (24–48 hours minimum). Make sure the envelopes are properly labelled with the genotype, plant number, and group.

 

  1. Weigh to determine the Dw at least 24 hours later.

 

  1. Calculate the RWC as (Jones 2007): Image

9.3.3) Rosette dehydration experiment (optional)

The monitoring of water loss of detached rosettes over a short period of time provides information about dehydration mechanisms. This is easily done by detaching rosettes and weighing them regularly over time to determine the dehydration rate. By plotting percentage of original weight vs time it is possible to determine whether a leaf regulates water flux through stomatal control (the initial phase of water loss) or cuticular evapo-transpiration (the second phase of water loss). Both processes may differ between the wild type and mutant plants.

Materials

Method

Scissors, plants, scale

  1. Cut a rosette from a well-watered (WW, control) plant and weigh it at regular intervals for two hours. The preferred intervals are: every 10 minutes for the first hour, and every 20 minutes for the second hour.

 

  1. Plot percentage of original weight vs time.

9.3.4) Gas exchange (optional)

If your plants are big enough (or still sufficiently alive) you may want to measure stomatal conductance during your drought experiment using the porometer, or LiCor if leaves are big enough and turgid. Consult with your supervisor. If possible, measure gas exchange on at least three plants per genotype per water treatment so that you can assess the effects of drought stress statistically. Refer to activities 7 and 8.

9.3.5) Harvest (optional)

Harvesting the above- and below-ground parts of your plants will enable you to do final growth measurements and determine leaf mass to area ratio (LMA) and root to shoot ratio. This optional activity can be performed on the well-watered plants or both.

Materials

Method

Plants, paper bags, labelling pen

  1. Remove above-ground parts at soil surface for three–five plants each of wild type and mutant.

 

  1. You may like to count leaves at this stage or measure the area of some or all leaves (see below).

 

  1. Separate any reproductive structures (bolt, flowers and siliques) and place in a labelled paper bag.

 

  1. Place rosette in a labelled paper bag.

Rubbish bin, trays or buckets of water, sieves

  1. Gently tip plant from pot and remove as much soil from roots as possible with your hands.

 

  1. Place roots in a tray of water and tease out all remaining soil. You may need to rinse the roots, or to change the water periodically. Make sure that soil does not go down the sink.

 

  1. Dispose of pots and soil as directed.

Drying oven, scales

  1. Place bags in drying oven (24–48 hours minimum) and return to weigh all parts.

9.3.6) Measuring leaf areas and LMA (optional)

The area and shape of individual leaves can be measured using photographs or scans of leaves.

Materials

Method

Scanner or digital camera, leaves

  1. Scan or photograph your leaves. Make sure leaves are labelled and each image has a scale marker on it.

 

  1. Determine leaf area using ImageJ software7.

Paper bag, labelling pen, scales

  1. Place leaves in labelled paper bag, dry as for 9.3.2, weigh to calculate LMA.

9.4) Expected outcome

For all plants grown at both conditions:

  1. Describe the phenotype of wild type and mutant plants grown under both conditions. When do plants start wilting? Are there pigmentation differences? Do the observations correlate with the findings in activities 4 and 5?
  2. Calculate and plot the RWC. Are there statistically significant differences? Remember, this is a factorial design so statistical tests will need to be two-way tests. See Appendix B.
  3. Are there any differences in final biomass (Dw)?
  4. How is the gs affected by drought? Are there any statistically significant differences?
  5. From harvest data, compare the growth of your plants with and without drought. Do the plants differ in total mass? Does relative allocation above and below ground differ between genotypes? Between drought treatments?
  6. Do leaf area and LMA differ?

 


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