Key words

1 Introduction

The first scientific observations of phototropic plant responses date back to the sixteenth century (reviewed in Ref. [1]). Darwin discovered that the perception of the stimulus and the site of action could be separated [2] which later led to the discovery of Auxin [3–5]. The responsible photoreceptors were identified with the help of molecular-genetics in the model plant Arabidopsis thaliana and later named after their major response: PHOTOTROPINS (phot) [6, 7]. Arabidopsis has two phots (phot1 and phot2) which are also involved in the regulation of chloroplast movement and stomatal opening. They are light-activated kinases. A sensory part with two LOV (light, oxygen, voltage) domains inactivates the kinase domain in the dark. After activation the sensory domain releases the kinase domain and phots can phosphorylate target proteins [8]. Phot1 regulates the phototropic response to a wide range of blue light intensities while phot2 mainly mediates the response to high-intensity blue light [9].

Other photoreceptors modulate phot mediated phototropism, mainly the blue light sensing CRYPTOCHROMEs (cry1 and cry2; [10]) as well as the red light sensing phytochromes, especially PHYTOCHROME A (phyA) [11–14]. While crys and phyA appear to enhance the phototropic response to blue light, cryptochromes also seem to be responsible for residual phototropism in the phot1phot2 double mutant [15]. Additionally phyA has been shown to mediate the enhancement of phototropism by a red light pretreatment [11, 16, 17]. Consequently a cry1cry2phyA triple mutant has a severely disturbed phototropic response [18]. Signaling factors for phototropism downstream of the phots have also been identified, among them NONPHOTOTROPIC HYPOCOTYL 3 (NPH3), ROOT PHOTOTROPISM 2 (RPT2) [19, 20], and the PHYTOCHROME KINASE SUBSTRATE (PKS) family with four members (PKS1–4). PKS1, PKS2, and PKS4 are associated with the plasma membrane and interact with phot1, phot2, and NPH3 [21–23]. PKS1, 2, and 4 as well as NPH3 have been shown to influence auxin distribution and auxin-dependent gene expression [24, 25]. Further downstream auxin transport facilitators are involved in phototropism. Among them are the PIN-FORMEDs (PINs), ATP-binding-cassette B-type transporter (ABCB) , and the AUXIN RESISTANT (AUX) 1—LIKE-AUX (LAX) family proteins [26–31].

Many different techniques have been described to analyze phototropism in Arabidopsis seedlings from which the growth on vertical oriented agar plates is the most common and easiest, and therefore this technique is described here. Others, like continuous video documentation of the seedlings [32] or microbeam irradiation on single seedlings [33] have also been used successfully to measure hypocotyl bending in unilateral illumination conditions. They require a more sophisticated setup and data management but deliver time resolved information. Nevertheless, for the routine test of few lines the technique described here is easy and requires no specific instruments or software. Results can be obtained in less than a week.

2 Materials

  1. 1.

    20–40 Arabidopsis seeds per line you want to investigate.

  2. 2.

    ½ MS solid medium: 2.15 g/l MS (Murashige–Skoog salts, without vitamins; see Note 1 ) in H2O (Milli-Q). Adjust pH to 5.7 with KOH, add 0.7 % agar, and autoclave. Pour in sterile square petri dishes (12.5 × 12.5 × 1.5 cm).

  3. 3.

    Sterilization solution: 2.5 % (v/v) sodium hypochlorite in H2O (Milli-Q) with 0.1 % (v/v) Triton X-100. Prepare fresh and be careful because the solution is quite corrosive.

  4. 4.

    Plating Solution: 0.1 % agarose (see Note 2 ) in H2O (Milli-Q), autoclaved.

  5. 5.

    A narrow band width light source (full width at half height; FWHH < 30 nm) with the option to adjust different fluence rates is necessary. Usually a slide projector with an interference filter can be used. Much better are high power LEDs that are easily available. For unilateral blue light we use 450 nm Luxeon high power LEDs (Roithner Lasertechnik, Austria), which are mounted on a 2-mm thick aluminum plate for cooling and connected to a power supply. Fluence rates between 1 and 100 μmol m−2 s−1 can be easily obtained by changing the current, and for lower fluence rates semitransparent paper is brought into the light path.

  6. 6.

    Black paper.

  7. 7.

    Book stands or black wooden boxes in which up to 6 square petri dishes will fit (inner dimensions 10 × 13 × 13 cm one side open).

  8. 8.

    Computer with the ImageJ software (National Institutes of Health) installed.

3 Methods

3.1 Seed Preparation

  1. 1.

    You will need 1–2 petri dishes per genetic line to investigate. Take the plates with the solidified media, turn them bottom-up and draw a line on the bottom of the petri dishes. The start point should be 5 mm before the top-midpoint of the plate and the end point 5 mm after the bottom midpoint of the plate if it lies on a table in front of you (Fig. 1a; see Note 3 ). Turn the plates back to the normal position (bottom-down).

    Fig. 1
    figure 1

    Schematic drawings of plate layout and light path to the plate stack. (a) View of open plate with the line on the bottom slightly rotated in relation to the center of the plate. Seeds are separately arranged on this line with a distance of 3–5 mm each. (b) Plate is closed and placed vertically so that the TOP (see also a) is on the upper side. Plates are stacked with black paper in between them as well as on the outside and on the back of the stack. The stack is slightly turned so that the light falls with a 0.5–1° angle on the agar surface

    Full size image
  2. 2.

    Sterilize the seeds by mixing them with 500 μl of sterilization solution in 1.5-ml reaction tubes and let the suspension incubate for 10 min at room temperature (see Note 4 ). Centrifuge briefly. From this point onwards work on a sterile bench. Discard the sterilization solution quickly and wash the seeds three times with 1 ml of sterile water. After the last wash, discard the water, add 100 μl of 0.1 % agarose, and mix.

  3. 3.

    Pipette some of the seeds in 0.1 % agarose on the drawn line on the MS medium (Fig. 1a). Move the single seeds with a sterile inoculation loop. Make sure that the distance between each seed is approximately 3–5 mm (see Note 5 ). Let the surface of the medium dry briefly on the clean bench so that the seeds do not shift when the plates are placed in the vertical position. Close the petri dishes and label them on the bottom with the genotype of the seeds they contain.

  4. 4.

    Stratify your seeds for 3–4 days wrapped in aluminum foil at 4 °C, already in the vertical position. Mark the top side of the plates.

3.2 Germination and Illumination

  1. 1.

    Unwrap the plates and expose 2 h to white light to synchronize germination.

  2. 2.

    Place the plates back in standing position and insert a black paper between each separate plate to minimize reflection. Also put a black paper at the back of the plate stack.

  3. 3.

    The plates should now be in a stack in which all the plates are standing, with black paper on both sides of the plates and on the back of the cube-like stack (Fig. 1b; see Note 6 ).

  4. 4.

    Let seeds germinate and the emerging seedlings grow negatively gravitropically for 2–3 days in darkness at 20–25 °C. Ideally, the plates should already be in front of the irradiation setup. The irradiation setup and position of the plate stack should be prepared in a dark room or in a completely dark cabinet.

  5. 5.

    After the germination and negative gravitropic growth, switch the light on to irradiate the seedlings from one side with blue light of the desired fluence rate for 12 h (see Note 7 ). The plates should be slightly moved out of the direct straight line of the light path so that the light falls obliquely onto the agar surface (Fig. 1b). Depending on the light source, different fluence rates can be adjusted in the light field by shading the front side of the petri dish cubes with semitransparent paper.

  6. 6.

    Document the phototropic bending angle by digital photography. Place a piece of millimeter paper next to the seedlings to obtain a scale.

3.3 Measuring the Bending Angle with ImageJ

  1. 1.

    Install ImageJ from http://rsb.info.nih.gov/ij/ and start the program.

  2. 2.

    Open your image: File>Open.

  3. 3.

    Make sure the plate is aligned with the border of the window: Image>Transform>Rotate.

  4. 4.

    Change the image to 8-bit greyscale: Image>Type> 8-bit.

  5. 5.

    Click on the angle-measuring button in the tools menu.

  6. 6.

    Click once on the base of the hypocotyl, once on the midpoint of the bend and last on the upper part of the hypocotyl.

  7. 7.

    Press M on the keyboard to save the measurement in the results windows.

  8. 8.

    Go back to step 6.

  9. 9.

    When finished with one biological sample, copy the data to Excel or a similar program to plot the angles (see Note 8 ).

4 Notes

  1. 1.

    Adjust the amount of MS salts according to the recommendations of the supplier. The number here refers to MS-salts from Genaxxon (Germany).

  2. 2.

    Agarose is used here and not agar. The low concentration agarose has a similar density as the seeds and allows them to float. This makes pipetting and manipulating the seeds much easier.

  3. 3.

    A slightly slanted line is intended so that the seedlings do not disturb each other when growing negatively gravitropically.

  4. 4.

    It is important not to exceed these 10 min because then the seeds suffer damage and germination rates drop. On the other hand, a much shorter time does not sterilize the surface sufficiently. Therefore 10 min should be observed closely (+/− 1 min).

  5. 5.

    This may be difficult at the beginning because the seeds tend to stick to each other in the drops of liquid. Therefore, less liquid is better. Try first to position a few seeds and when you are done, take more out of the tube onto the agar surface.

  6. 6.

    Care should be taken that the plates are exactly upright. This can be achieved by constructing wooden boxes or by using bookstands on both sides of the upright stack.

  7. 7.

    For standard tests a fluence rate of 1 μmol m−2 s−1 should result in a good response of the wild type. Fluence rates should be determined with a PAR meter or similar device at the point where the seedlings will be positioned.

  8. 8.

    Usually it is not conclusive to take a mean value of the angles, especially if plants are not behaving normally (for instance showing a positive or negative reaction towards the light source). It therefore is better to take classes of angles (0–10°, 11–20° etc.) and sort all the measured values into these classes, so that a table is generated which comprises numbers of seedlings per class. This table needs to be normalized to the number of seedlings by translating the numbers per class to percentage of all seedlings per class. This dataset contains more information than the mean (Fig. 2).

    Fig. 2
    figure 2

    Different presentations of the same bending angle data. Three artificial data sets, representing a normal distribution, a wide distribution, and an avoidance component in the bending response were plotted with three different methods. (a) An average does not differentiate between mutant1 and mutant2 (positive and negative phototropism) nor does it reflect the avoidance reaction. Error bars represent standard errors of the mean. (b) All seedlings were sorted into classes and normalized to the total amount of seedlings from each genotype. This is a much better representation of the response and the differences between the different genotypes. In (c) the same data were converted into circular histograms (wuschel-diagrams). Here the length of a line represents the percentage of seedlings in this specific angle class. This diagram, also based on the same data set as in b, is more informative. The light was coming from the right hand side as indicated by the arrow and the scale bar is 10 % of the seedlings represented

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