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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.

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 which is always good) 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 specificty 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.

Calibration curve with synthetic auxin in LB

Colour change from yellow to red with increasing auxin concentrations

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.

Results from salkowski assay with cell filtrate (add pore size)

2. HPLC

3. Testing the effect of auxin on Arabidopsis

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 will use DR5:GFP and DR5:3XVENUS plants that respond to auxin by expression of GFP and YFP, respectively, to look at the plant response to synthetic auxin and later bacteria-secreted auxin. The DR5 plant lines respond to auxin exposure by expressing GFP and YFP, respectively. 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 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 will be supplying the plants with synthetic auxin and observe the differences in growth and (root) morphology due to differential concentrations of the hormone. In later stages of the project, this will be followed by exposing the plants to E. coli cells expressing auxin.

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.

The inhibition of root elongation at higher concentrations of auxin

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 photoradiography. The root length data collected were analyzed using ImageJ comparing to a reference.

Figure : the effect of different IAA concentrations from 0.1 nM, 10nM, 1 µM, and 0.1 mM on root growth. (left) The top panel shows the effects of root growth by 0.1 nM IAA and bottom panel for 10 nM (middle) The top panel shows the effects of root growth by 1 microM IAA and bottom panel for 0.1 mM (right)

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

Linear graph showing root length decreases logarithmicly when increasing auxin concentrations in liquid media

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.

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 photooxidation 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 photoradiography. 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 (0M)were applied at 2 cm, 4 cm, 6 cm, 8 cm, 10 cm away from the seeds (right)

Figure : phytogel plates applied with IAA concentrations of 10 µM (top left), 1 µM (top right), 0.1 µM (bottom left)and control (0M) (bottom right)

The average root length at each position from the mentioned positions were plotted as a graph to compare the effect in root length in different IAA concentrations and to find the optimal distance to apply IAA.

Curves showing the decrease of root length ratios on increasing auxin concentrations in root split experiment

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 nearer concentration. Also the results suggest the inhibiting effect from IAA concentrations of 1 µM and 10 µM (higher extent in the latter) since root length decrease in nearer distance but become increasing as the concentrations are diluted away by further distances.

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 photoradiography. The maximal root length data collected were analyzed using image J program by comparing with the reference. The maximum root length was plotted for each IAA concentration.

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.

Figure : The split plate applied with IAA concentration of 10 µM (top left), 1 µM (top right), 0.1 µM (bottom left) and 0.1 nM (bottom right)

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

Curves showing effect of root lengths on different auxin concentrations in phytogel

The ratio is shown to fit logaritmicly with increasing IAA concentrations. However, the inhibiting effect fpound 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. Confocal imaging

Confocal imaging was used to image our plants for several experiments. Confocal microscopy is especially useful for this purpose as it makes three-dimensional imaging of samples possible and lasers with differing wavelengths can be used to excite fluorescent samples in parallel.

We used this type of imaging to observe the effect of auxin on the DR5 mutants. Plants incubated with 0.1mM of IAA showed strongly enhanced lateral root growth but also stunted growth.

A Z stack through an Arabidopsis root tip incubated with 0.1mM indole 2-acetic acid.

This did not occur at lower concentrations. However, fluorescence was still clearly visible:

Root of A. thaliana seedling incubated with 1uM indole 3-acetic acid.

At even lower concentrations, fluorescence was much weaker:

Incubated at 0.01nM.

Soil erosion experiment

In order to see which root architecture, the length and the dispersion, is the best to hold up the soil to prevent soil erosion and retain the moisture inside the soil. We then varied 8 concentrations from 0.01 pM, 0.1 pM, 1 pM, 10 pM, 0.1 nM, 10nM, 0.001mM, and 0.1mM as the same as the effect of different IAA concentrations experiment. These different IAA concentrations allow the roots to show different architectures. The experiments were planned up as shown in the diagrams below.
- The material for making the slope was the 20 cm x 10 cm pots placing on top of the 30 degree slope.
- The soil chosen is called M2 which composes of organic compost without any gravel to eliminate the error from different mixtures which contributes to different soil contents. The soil immitates normal
- The pressure of water is kept constant by the adaptable shower head which immitates the average speed of rainfall (9.8 m/s)

The experiment is set up where 18 (3 columns at 10 cm side x 6 rows on 20 cm side) arabidopsis were seeded on the pots which represented each concentration. At each concentration, the experiment was repeated 3 times. These altogether with 2 controls where no IAA was watered in the first one and no Arabidopsis was seeded in the second one, result in 30 pots in total. Estimately 12.5 kg of soil (1/4 of 50kg M2) is used for the experiment. The soil was pretreated with 2 litre of 1mM fungicide solution and left dryout for 1 day. It was then seperated to approximately 400g on each pot and was watered each with 25ml of water to moist up the soil conditions for Arabidopsis seeding. A small hole was digged 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 in a 2 days afterwards. Each pot was watered with 25 ml of auxin concentrations mentioned above.

The data on the number of plants grow with less 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.

After the plants have been grown for 3 and a half week. The plant was watered with prepared concentrations. Then, after 10 minutes 10 cm³ of soil at a region between 4 plant roots was collected for a the wet mass weight. The soil was then incubated to get the dry mass. The different 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 retention was repeated. The least differences from water hold up between 10 minutes after watering and 2 days after, the more water retention the soil is.

To see how rooting could prevent soil erosion, the experiment is set up as picture below, where the soil basket was cut opened at one end.

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.