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From 2011.igem.org

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Module 2: Auxin Xpress

Auxin, or Indole 3-acetic acid (IAA), is a plant growth hormone which is produced by several soil bacteria. We have taken the genes encoding the IAA-producing pathway from Pseudomonas savastanoi and expressed them in Escherichia coli. Following chemotaxis towards the roots and uptake by the Phyto Route module, IAA expression will promote root growth with the aim of improving soil stability.

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Testing

How much auxin will we be producing? More importantly, has the module actually worked?! This chapter will look into the methods that we have decided to use in order to measure the amount of auxin in a solution. We have decided to use qualitative methods such as the Salkowski reagent (changes colour) as well as quantitative methods such as HPLC and GC-MS for more accurate results. We are also doing experiments on Arabidopsis root length with synthetic auxin.

1. Salkowski assay

Salkowski is a colourimetric assay that detects indole-3-acetic acid with high specificity among other indoles. This standard assay is the simplest way to determine whether there is auxin present in solution. First we created a standard curve with increasing auxin concentration in LB broth using synthetic auxin.

The aim of this experiment was to determine whether our system is working and roughly how much IAA we are producing when compared to our control cells.

First we set up the assay with synthetic IAA dissolved in LB to make a standard curve that we could use as a reference to convert OD measurements to concentration when doing the assay with auxin producing cells. (figs. 1&2)

Figure 1: Standard curve of Salkowski assay made with synthetic auxin in LB

Figure 2: Cuvettes used to measure OD for the standard curve. As auxin concentration increases, the solution progresses towards red.

We did preliminary tests with our auxin producing cultures with a spectrophotometer. Once auxin presence was confirmed by colour change, we set up a more thorough assay with a BMG Omega plate reader. The first assay we did was with E. coli DH5 (Figs. 3&4), the results of which were positive for our auxin construct. The auxin producing E. coli were producing approximately 55 uM of IAA. From modelling, we have determined that our construct would be able to produce 72.25 uM IAA, which shows that we were are in the correct order of magnitude.

Figure 3: Results from trial 1 of Salkowski assay with cell filtrate of auxin producing E. coli DH5α. Filtered through a 0.2 µm pore filter

Figure 4. Visual results correlating with OD measurements. The eppendorf on the right contains auxin producing E. coli DH5α and the eppendorf on the left contains control E. coli DH5α.

Due to inconsistent results with colour change on various repeats, we redid the Salkowski assay with repeats using different controls. We tested different growth media, incubation temperature, tryptophan concentration and light exposure to optimize IAA production.

2. HPLC

3. Testing the effect of auxin on Arabidopsis - imaging fluorescent reporter lines

We are not only interested in constructing the auxin-producing pathway in our bacteria but we also want to investigate what effect the auxin has on plants to verify our assumptions about indole 3-acetic acid (IAA)'s effects. This will help us with the human practices aspect of our project and it will also provide a good assay for the functionality of auxin-secreting bacteria.

To observe how indole 3-acetic acid influences plants, we will be working with the plant model organism Arabidopsis thaliana. Arabidopsis is well-established for research into plant biology and researchers have established lines that respond to auxin exposure by expressing reporter genes, which are particularly useful for our project.

We used DR5:3XVENUS plants that respond to auxin by expression of YFP to look at the plant response to synthetic auxin and bacteria-secreted auxin. This will allow us to monitor how much auxin is taken up and which cells respond to it. We will be using confocal microscopy to image and evaluate the relative strength of fluorescence expressed by the plant. This will act as an indirect reporter on the auxin concentration supplied as it relies on the plant expressing fluorescence in response to stimulation by the hormone.

Initially, we supplied the plants with synthetic auxin and observe the differences in growth and (root) morphology due to differential concentrations of the hormone.

We exposed Arabidopsis seedlings to different concentrations of IAA to observe its effect on the plant. For this, we used DR5:3VENUS, a mutant that responds to auxins by expressing the fluorescent protein YFP. Confocal imaging was used to image our plants for this experiment. This type of imaging is especially useful as it makes three-dimensional imaging of samples possible thanks to precise imaging of individual points.

Plants incubated with 0.1mM of IAA showed strongly enhanced lateral root growth but also stunted growth. This does not come as a surprise as concentrations this high are usually considered detrimental for the plant.

Video 1. Z-stack imaging of A. thaliana roots exposed to 0.1mM IAA (imaging by ICL iGEM 2011)

At lower concentrations, stunted growth was not observed. However, root tip cells were very brightly fluorescent and increased root branching could be observed.

Video 2. Root of A. thaliana seedling incubated with 1uM indole 3-acetic acid (imaging by ICL iGEM 2011).

At even lower concentrations, fluorescence was almost impossible to detect and much less branching of the root can be observed.

Video 3. Response of the roots to 0.01nM IAA (Imaging by ICL iGEM 2011).

Once we had engineered our bacteria to express IAA, we repeated this experiment with the bacteria supplying the plants with auxin. While plants supplied with no bacteria were glowing very brightly in response to the IAA produced by the roots themselves, the roots supplied with bacteria unable to express auxin only showed very weak fluorescence. Plants supplied with auxin-expressing bacteria were brighter than any of the two controls.

Video 4. Response of the roots to IAA-expressing bacteria.

It therefore seems as though bacteria normally suppress production of IAA in root tips. However, the bacteria we have engineered to express IAA produce enough of the compound to not only overcome this limitation but also to cause higher expression of the reporter gene

4. Testing it effect of IAA on Arabidopsis - observing root length

4.1 The promotion and the inhibition of root elongation at different concentrations of auxin in liquid media

To look at the inhibiting effect of auxin on plants, we supplied different indole 3-acetic acid concentrations to Arabidopsis seedlings in liquid culture.

Si modelled the concentration of auxin secreted by our bacteria to be 10mM. However Joseph et al. 1995 state that the optimal auxin concentration lies in between 0.5 µM - 20 µM. Accordingly, we used concentrations starting from 10mM to 0.1nM to test the effect of different auxin concentrations on the length of the roots and their branching. We made the auxin concentrations by serial dilution and added 10ml of concentrated auxin solution to 100ml of half-MS media each. Twenty-five seeds were added to each flask. The seeds were incubated at 23°C and wrapped in aluminium foil to allow the plants to germinate in the dark. They will be allowed to grow in the light in 3 days' time. This follows a protocol described by Joseph et al. (1995).

After 3 days in light, 4 plants from each flask were collected and imaged with autoradiography. The root length data collected were analyzed using ImageJ comparing to a reference.

The average root length are plotted into the graph to illustrate the inhibiting effect of root elongation, which logarithmically fit with the increasing IAA concentrations.

Even though the result suggest here is not in consensus with the literature. Therefore, in order to see the effect of auxin in root length promotion, we extend the experiment further down to the concentrations of 0.01 pM

References:
[1] Joseph J. King, Dennis P. Stimart, Roxanne H. Fisher, and Anthony B. Bleecker, ''A mutation altering auxin homeostasis and plant morphology in Arabidopsis'', The plant cell, Vol.7(December 1995), 2023-2037.

4.2 Root growth differs according to the distance of indole 3-acetic acid.

Looking at the effect of auxin on the roots also involves observing its effect on phytogels. On these gels, individual seedlings grow horizontally into a gel containing plant nutrients. These gels enable us to supply the plant with auxin at set distances from the seedling itself. Observing these effects is especially in case our bacteria do not get taken up into roots in nature, which has yet to be investigated. We are using DR:3VENUS seeds. These germinate into plants whose roots respond to auxin uptake by expressing YFP. We hope to be able to get an estimate of the effect of auxin by comparing intensities of fluorescence across plants supplied with different concentrations of auxin.

Si corrected the estimated IAA secretion of our bacteria to 0.0001 to 0.01 mM. Accordingly, we set up the auxin concentration gradient experiment with 0.0001, 0.001 and 0.01 mM of IAA. We injected a small volume of IAA dissolved in 70% ethanol at set distances from seeds, which were subsequently put onto the gel. Five replicates were set up for each concentration. On each plate, the seed seeds are sown at distances of 2,4,6,8, and 10 cm from the point where IAA is applied. Roots grew perpendicular to the line on which IAA and seeds are applied. The plates were kept in low light for 3 days in order to prevent photo-oxidation of auxin and also in 4 C to simulate the winter hibernation. The plates are later put in light for another 6 days to see root growth.

After 6 days, the phytogel plates were imaged with autoradiography. The root length data collected were analyzed using ImageJ comparing to a reference. The data collected was plotted to see the optimal root length for each IAA concentration.

The examples of phytogel plates where IAA with concentrations of 10 µM, 1 µM, 0.1 µM and control (0 M) were applied at 2 cm, 4 cm, 6 cm, 8 cm, 10 cm away from the seeds from left to right.

The average root length at each distance from the IAA applying positions were plotted as a graph to compare the effect of root length in different IAA concentrations. The largest root length from 5 distances could tell the optimal IAA concentrations required to apply for particular distances. The information could help the modeller and the auxin team predicting the strength of the promoter and the regulation required for the bacteria to express IAA at different distances from the plants.

Here, the results show the optimal distances away from seedlings in the range of 2-10 cm listing at below 0.1 µM : 2 cm, 1 µM : 6-8 cm, 10 µM : 6-10 cm. The results support the root length promoting effect by 0.1 µM of IAA since the root grows more in the nearer proximity to the IAA applying position. Also the results suggest the inhibiting effect from IAA concentrations of 1 µM and 10 µM (higher extent in the latter) since root length decreases in shorter distance from applying position but increases as the concentrations are diluted by longer distances.

4.3 The split root experiment

To visually compare the difference of the root growth between the applying and non applying of IAA in the same plant, we set up a split root experiment (which had been recommended to us by Dr Alex Milcu). This experiment extends beyond the scope of normal controls as the same plant is subjected to two different treatments. We supplied the following concentrations of IAA to one half of the roots: 0 µM (control), 0.1 µM, 1 µM and 10 µM, while the other half was grown in phytogel containing no IAA. 3 replicates were set up for each concentration. 7-day old seedling of A thaliana DR:3VENUS were used for this set up. This strain responds to auxin by expressing YFP. The plates are sealed and kept in the incubating room for 2 weeks in order to observe the length of the grown root.

After 2 weeks, the phytogel plates were imaged with autoradiography. The maximal root length data collected were analyzed using image J program by comparing with the reference.

Below are remaining split root plates which were not infected by the fungus. The split plate contains the control half (on the right side of the plate) and a half where different concentrations of IAA are applied.

The length of the roots analysed using ImageJ are written on the table, which could be used to calculated the ratio between the IAA applied samples and the controls.

The ratios between the IAA samples and the control at each concentration were calculated and plotted into a graph below

The ratio is shown to fit logarithmically with increasing IAA concentrations. However, the inhibiting effect pound in phytogel is higher than the liquid medium. We are then carrying on the experiment with lower IAA concentrations to observe the positive effect of IAA in root length.

4.4 Soil erosion experiment

In order to determine the best root architecture, in terms of length and dispersion, for holding up of the soil in order to prevent soil erosion and retain moisture inside the soil, we used 8 concentrations: 0.01 pM, 0.1 pM, 1 pM, 10 pM, 0.1 nM, 10nM, 0.001mM, and 0.1mM (same as the effect of different IAA concentrations experiment). These different IAA concentrations allow the roots to show different architectures.
The experiment was set up with 18 (3 columns at 10 cm side x 6 rows on 20 cm side) Arabidopsis were seeded in 3 rectangular pots which represented each concentration pf IAA. Together with 2 controls (one where no IAA was watered and one where no Arabidopsis was seeded), 30 pots in total. Approximately 12.5 kg of soil (1/4 of 50 kg M2) was used for the experiment. The soil was pretreated with 2 L of 1mM fungicide solution and left to dry out for 1 day. It was then separated to approximately 400g in each pot and was watered each day with 25ml of water, in order to moist up the soil conditions for Arabidopsis seedling. A small hole was dug onto each seeding place to fix the position of the seeds. Arabidopsis was seeded with the pattern already described. For the first 3 days, the seeds were watered everyday at 4 pm to promote the growth and the watering was decreased to once every 2 days afterward. Each pot was watered with 25 ml of auxin concentrations mentioned above. The plant growth and the number of leaves were noted every 3 days as below.

The data on the number of plants grow with fewer or equal to 2 leaves (shown in black) and ones with more than 2 leaves (shown in red) watered with different IAA concentrations are tabulated below.

As expected, the plants watered with IAA concentrations ranges of 10 pM-10 nM emerge first from the ground and produce more number of leaves than the others.

After the plants were grown for 4 weeks. At 10 minutes after final watering, 10 cm³ of soil at a region between 4 plant roots was collected for measuring wet mass. The soil was then incubated for 1 day to get rid of water to achieve dry mass. The difference between the wet and the dry mass was the water hold up in the soil. The plants were left 2 days with no watering and the same procedure to measure water hold up was repeated. The smaller the difference of water retained between 10 min and 2 days after watering, the more water was retained by the soil.

To see how rooting could prevent soil erosion. The experiments were carried out as shown in the diagrams below.

- the soil basket was cut opened at one end to allow water with eluded soil to flow into the basin. - The slopes were made by placing 20 cm x 10 cm baskets on top of the 30 degree slope.
- The soil chosen is called M2 which composes of organic compost and sand without any gravel to eliminate the error from different mixtures contributing to different soil contents. M2 imitates the normal soil found in degrading area.
- The pressure of water is kept constant by the adaptable shower head which imitates the average speed of rainfall (9.8 m/s). The shower honk is placed at 90 degree to the floor 30 cm above the top of the plant pot.

The eroded soil collected from the basin was incubated to measure the dry mass. The higher the dry mass collected, the more soil is eroded.

References:
[1] Stijn Spaepen, Jos Vanderleyden, and Roseline Remans, “Indole-3-acetic acid in microbial and microorganism-plant signaling,” FEMS Microbiology Reviews 31, no. 4 (July 2007): 425-448.