Hormone Synthesis in Chlamydomonas reinhardtii

A Novel Approach to Determine the Hormone Synthesis Pathways in Chlamydomonas

reinhardtii
Lamees Al Alshaikh
Dhahran Ahliyya Schools

Abstract
The identification of the family of plant hormones called strigolactones as the chemical signal
that represses plant tillering and promotes the formation of longer roots opened the door for
the elucidation of their functionality to incorporate them in genome engineering of crops and
combating parasitic weeds. To achieve a better understanding of these hormones, this study
focuses on identifying similar pathways in the lower plant and model organism,
Chlamydamonas reinhardtii, a unicellular green algae. This was approached through
examining the function of genes producing analogous enzymes to those known in Oryza
sativa, through reactions with various isomers of -carotene. High Performance Liquid
Chromatography (HPLC) was used to identify downstream products. The reactions of a D27
homolog in C. reinhardtii was found to be analogous to that of D27 in Oryza sativa. A new
compound in carotenoid pathways was determined to be the product of the reaction between
the substrate 9-cis--carotene with analogous CCD7 from C. reinhardtii. The identification of
the D27 homolog as a part of a similar pathway in Oryza sativa is a key step in elucidating
the full pathway in C. reinhardtii. The product of the analogous CCD7 will also be examined
and analyzed to provide a new pathway for strigolactones synthesis or perhaps an entirely
new family of hormones.

Hormone Synthesis in Chlamydomonas reinhardtii

1.0 Introduction:
Hormones regulate the various stages of growth in plants, from germination through root
elongation and flowering (Davies, 1987). Greater understanding of how these hormones are
synthesized and how they regulate stages of growth and development would allow us to
modify crops for stressful environments. In order to understand the basics of how plant
hormones are synthesized, investigations of

their synthesis pathways is necessary. An

important example of these are the strigolactone hormone synthesis pathways in C.

reinhardtii, which is a model unicellular green algae that can habitat the soil and water
bodies.
Strigolactones are hormone derivatives of carotenoids that are synthesized in plants. One of
their roles is promoting the establishment of the symbiotic relationship of plants with
mycorrhizal fungi (Smith, 2014). The mutualism between the plants and the fungi is initiated
when the plant provides the fungi with energy produced in photosynthesis and the fungi
provides nutrients and minerals from the soil to the plants. Not only does the secretion of
strigolactones trigger this symbiotic relationship, it also induces seed germination of root
parasitic plants. While this induction may seem harmful, it can be manipulated and used to
combat parasitic weeds for better crop yields.
In higher plants, -carotene is a precursor for the synthesis and secretion of strigolactones. An
examination of the algae, C. reinhardtii, is necessary to determine whether or not it has the
same pathways as higher plants i.e. forms hormones from carotenoids (Al Babili and Cabrero,
2014). Previous literature used Carotenoids cleavage dioxygenases (CCDs), which are
enzymes that cut C-C double bonds of carotenoid by inserting a dioxygen , to synthesize
strigolactones in vitro (Alder et al., 2012). For this reason, a similar examination of a family
of CCDs enzymes was done in C. reinhardtii. This would also allow further understanding of

the evolutionary development of strigolactones from lower plants such as C. reinhardtii to

higher plants such as Arabidopsis thaliana and Oryza sativa.
It is important to find similar pathways in lower plants because the pathways in higher plants
are much more complex and harder to study. By going back in the evolutionary chain,
hormone synthesis reactions that happen inside C. reinhardtii are much easier to examine and
analyze than those in higher plants (Al Babili and Cabrero, 2014).
It is of great significance to understand how strigolactones function and to discover new
hormones that have similar functions.

If C. reinhardtii secretes strigolactones or

strigolactone-like hormones to induce suicidal germination of seeds of parasitic plants (Al

Babili and Cabrero, 2014), then this could provide us with tools to prevent parasitic weed
growth and propagation. Hence, prior to planting the intended crop, suicidal germination
could be induced, causing the parasite seeds to germinate and the parasite to eventually die of
the lack of a host plant for the parasitic plant to live off of. Using this method, the soil would
be sterile of any parasitic seeds and parasitic weeds will not affect commercially important
crops. These are just a few examples of how the identification of these hormones is relevant
to improving our capacity to produce food. However, this is not the only function of
strigolactones. In fact, they have many functions including the regulation of plant growth and
the stabilization of the symbiotic relationship between algae and fungi (Smith, 2014).
Strigolactones also play an important role in plant growth regulation as they send chemical
signals inhibiting tillering of secondary shoots. Instead, the growth energy is used to induce
the thickening of the stem, and promote the development of lateral roots. This way the plant
is more capable of scavenging for nutrients in the soil, while limiting the loss of resources
that would have been otherwise consumed by the unnecessary secondary tillers. This ties
back to the symbiotic relationship between plants and arbuscular mycorrhizae; where this

Hormone Synthesis in Chlamydomonas reinhardtii

relationship provides the plant with even more nutrients, explaining why natural
strigolactones are secreted at higher levels when nutrients are scarce in the soil (Smith, 2014).

2.0 Methodology:
2.1 Identification of potential strigolactone synthesis enzymes
In order to identify enzymes in C. reinhardtii that are similar to enzymes found in higher
plants, we used Basic Local Alignment Search Tool (BLAST). Sequences of four enzymes
found in higher plants (CCD7, CCD8, D27, and NCED) were compared to that of C.
reinhardtii. Several similar enzymes were found for each enzyme. However, we will be
examining the most analogous one.
2.2 Cloning:
Following the sequence alignment of the The genes of interest, CCD7-L1, CCD8-L1, D27L1, and NCED-L1 were ordered from GenScript, and gel purification was used to separate
the genes from the Puc57. EcoRI was the enzyme used for the digestion of the new pThio
vectors, then each gene was ligated its new vector. Because only one restriction enzyme was
used for the digestion of the genes and pThio, the gene could have been inserted in either
direction. For this reason, a confirmation digest was performed using the following enzymes:
SmaI was chosen for CCD7, SacI for CCD8 and NCED, and RsaI for D27.
2.3 In vivo assays:
In vivo is when the enzymes and substrates are tested within biological entities. For the in
vivo, competent E. coli strains were engineered to produce -carotene, the substrate. Cloned
pThio vectors were used; each vector contained one of the four genes encoding for the
enzymes of interest (CCD7, CCD8, D27, and NCED).
Following cloning was the transformation of the cloned vectors into the -carotene producing
bacteria. In order to do that, 150ng of each cloned vector was transformed into 50l of
previously prepared -carotene producing chemically competent cells by incubating them for

30 minutes on ice. This was followed by a 30 second heat shock in a 42 o C water bath, then
incubation on ice for 2 minutes. After that, 250l of Super Optimal broth with Catabolite

Figure 2.2 Colonies of CCD7 expressing bacteria on Agar plates.

Kanamycin and ampicillin agar plates under a sterile hood ready to be inoculated as part of the second day of a
repression (SOC), a liquid nutrient, was added in a 1.5mL eppendorf tube, and left shaking at
37o C for 1 hour. Lastly, Agar plates previously prepared with 100ng/uL ampicillin and
50ng/uL kanamycin were aseptically inoculated with 150l from each sample, and left to
incubate at 37o C overnight (16-20 hours).
The next day, a colony from each plate was inoculated in a loosely capped test tube
containing 3mL 2YT media and 50ng/L kanamycin + 100ng/L ampicillin. This was
incubated overnight shaking at 37o C in the dark. After that, 1 ml of the culture was
inoculated in 100 ml 2YT media with no antibiotic. This was incubated for approximately 4
hours at 28o C in the dark until the OD600 reaches 0.5 - 0.6, which was determined using a
spectrophotometer.

Figure 2.3 Arabinose induction visualization.

Arabinose binds to the repressor removing it with it, and then RNA polymerase begins the synthesis of the enzy

Hormone Synthesis in Chlamydomonas reinhardtii

When the optical density reached 0.5, arabinose was added to the culture at a final
concentration of 0.2% w/v (Figure 2.3), and then incubated overnight at 28 o C in the dark.
This step is called induction. Since induction is what causes the expression of the enzyme,
this step is very critical and should be done exactly when the bacteria reach the optimum
growth phase. During overnight incubation, and after the enzyme is secreted, the enzyme
reacted with the substrate produced by the bacteria.
Following incubation the product of the reaction between the enzyme and the substrate were
extracted. The samples were first centrifuged at a speed of 4000 rpm at 4 o C for 15 minutes.
The supernatant was then discarded and the pellets were immediately placed in the -80 o C
freezer ready for extraction. The samples were then taken out of the freezer and 4 volumes of
HPLC grade acetone was added to the sample. In order to run the samples in HPLC, the
products must be extracted via sonication to break the cell membranes. The samples were
then centrifuged for five minutes at 13, 000 rpm and the supernatant was filtered using a
0.2nm filter.

Figure 2.4 Pellet of enzymes reacted with -carotene after centrifugation.

Pellets of the final product of -carotene with the control and the four enzymes of interest extracted from the bacteria. The

The samples were then dried in a vacuum centrifuge then resuspended in 40L of HPLC
grade methanol, and loaded in HPLC.
2.4 In vitro assays:
Each vector cloned with the genes referred to in the in vivo was inserted into a BL21
competent cell using the same method previously describes in section 2.2. Following this
transformation is the inoculation of transformed bacteria in preparation for the induction. The

same methods used for the in vivo (section 2.3) were used for the in vitro with two variations:
the inoculation in 100mL of 2YT becomes 50mL of 2YT and the incubation time after
induction was specifically four hours.
Following induction was extraction of the enzyme from the cells, which was done for a
similar purpose to that of the extraction in the in vivo. Because BL21 does not produce carotene, the extraction was only of the active enzyme rather than the product of the enzyme
and the substrate. For this reason the extraction procedure was slightly different.
In order to perform the extraction, a lysis buffer was prepared which consists of 500L PBS
PH8.0, 100L of 1mM DTT, 0.1% Triton X-100, 300l water per assay.
First the sample was transferred in a 50mL falcon tube and was then centrifuged for 15
minutes, 4000 rpm at 4o C. After that, the supernatant was discarded and the sample was
dried. It was resuspended in 1mL of lysis buffer. This was then followed by the addition of
10L of lysozyme [1mg/mL] and was incubated for 30 minutes on ice. The sample was then
sonicated 3 times for 5 seconds with a frequency of 30kHz, and centrifuged to isolate the
proteins from cell debris.
The substrates, in our case the carotenoids, were 10'-apo--carotenal, 9-cis--carotene, alltrans -carotene, and Zeaxanthin. Each one was prepared at a specific concentration and
volume of 60M in 200L. The dilution that needs to be made was calculated, then the
necessary volume of the carotenoid from the stock concentration were added to a 1.5mL
eppendorf. Then the samples were dried in a vacuum centrifuge. The dried samples were
then resuspended in 50L of pure ethanol, followed by addition of 50L of 0.4% Triton X100. The sample was dried again for it to be used in the in-vitro assay.
An assay buffer was prepared, which consisted of 500L of 200mM Hepes pH 8.0, 10L
2mM TCEP, 10L of 0.4mM FeSO4, 60L [2mg/mL] catalase, and 420L water per 1mL of
assay buffer.

Hormone Synthesis in Chlamydomonas reinhardtii

The dried carotenoids were first resuspended in 50L of water. Then 100L of the assay
buffer was added along with 50L of the crude protein prepared earlier. These samples were
then incubated shaking at 28o C in the dark for a time period of up to 20 hours.
After this incubation the final extraction and filtration of the product of the enzyme and the
substrate was executed. To extract the final product, 400L of acetone was added to each
sample, vortexed, and then sonicated for 3 seconds 3 times. After that, 800L of Petroleum
ether:Diethyl ether 1:4 was added, vortexed, and then centrifuged for 30 seconds. The colored
liquid containing the products was then extracted, filtered using a 0.2nm filter, and
transferred to a new 1.5mL eppendorf tube. The samples were then dried in a vacuum
centrifuge. Once they were completely dried, they were resuspended in 50L of methanol and
ready to be run in the HPLC.
2.5 HPLC Analysis
Both the in vitro and in vivo samples were run through HPLC for around 30 minutes. The
products were then separated based on the size and properties of each compound in the
product. The settings of the HPLC used were as follows:
1. Flow: 0.600 ml/min.
3. Wash volume: 100L.
5. Column oven: 30o C.

2. Eluents: 100% Methyl tert-Butyl Ether.

4. Tray temp: 4o C.
6. Wavelength range: 300-500nm.

Figure 1.1 Sketch of the inner parts of High Performance Liquid Chromatography.
This is the tool for analysis of the final products obtained from our experiments. The separation of compounds b

10

3.0 Results:

Figure 3.1 Analysis of in-vivo incubations of -carotene with CrD27, CrCCD7, CrCCD8, CrNCED.

(CO1) Control graph from left to right displays three peaks of 15-cis--carotene, all-trans--carotene, 9-cis--carotene. (D

Hormone Synthesis in Chlamydomonas reinhardtii

11

Figure 3.2 Spectrum of the three peaks found in each graph in Figure 3.1.
The double shouldered shape of the graphs prove the belonging of each peak to the -carotene family. The loca

The E. coli strain that was engineered to produce -carotene produced two other isomers of
the carotenoid, allowing for more than one enzyme to react. D27-L1 of C. reinhardtii cleaved
all-trans--carotene into 9-cis--carotene decreasing the second peak relative to the third
peak, and resulting in the concentrations of all-trans--carotene and 9-cis--carotene
equilibrating (Figure 3.1, D27). The 15-cis--carotene isomer was not affected in the reaction
with D27-L1; the first peak did not change (Figure 3.1, D27).

12

CCD7-L1 cleaved 9-cis--carotene into a compound that was not yet identified in a
carotenoid pathway before (Figure 3.3). The third peak in Figure 3.1 relatively decreased and
an unknown compound eluted in the first two minutes (Figure 3.3).

Figure 3.3 Unidentified compound produced by -carotene synthesizing E. coli expressing CCD7-L1.

NCED-L1 appears to have a similar pattern as its assumed analog CCD8-L1 shown in their
corresponding graphs in Figure 3.1. as they both cleaved 9-cis--carotene and 15-cis-carotene while the all-trans--carotene remained unaffected.

4.0 Discussion:
D27-L1 in C. reinhardtii appears to have the same enzymatic function as D27 in Oryza
sativa, CCD7-L1 cleaves 9-cis--carotene, similar D27 in Oryza sativa does. However, the
products of this cleavage are not -ionone and 10'-apo--carotenal as predicted. Instead, in
the in vivo assay an unknown product has been identified that has not yet been characterized
in the carotenoid pathway. This unknown product requires further analysis to be understood
completely. Regardless, this product could be part of a different pathway producing a
different hormone, or it could be producing strigolacone-like hormones but simply through an

Hormone Synthesis in Chlamydomonas reinhardtii

13

alternative pathway. Since strigolactones have different synthesis pathways in different plants
it is very likely that this is a detour leading to the synthesis of similar hormones.
Originally discovered in the carlactone pathway of Oryza sativa, CCD8 functions in sequence
with CCD7 (Alder et al., 2012). We have yet to express both CCD7 and CCD8 in the same
bacterial strain, therefore, the products were not expected from CCD8 or its analog NCED.

Figure 4.1 In vitro incubation of both control and CrCCD8 with 10'-apo--carotenal.
CCD8 cleaved 10'-apo--carotenal into retinal. (A) shows the spectrum of the large peak between the retention

This does not mean that there will be no reaction between the enzyme and the available
substrates because regardless of its assumed function, it still remains a carotenoid cleavage
enzyme. The reason CCD8 acts on a product of CCD7 rather than 9-cis--carotene in Oryza
sativa is because CCD7 is more active than CCD8. However, in the experiment conducted
CCD7-L1 and NCED were only provided with 9-cis--carotene, rather than -ionone and 10'apo--carotenal, the products of CCD7s cleavage. This caused the only activity of both
enzymes to be cleaving 9-cis--carotene. Regardless, CrCCD8 could still be similar to that of
Oryza sativa. While CCD8s function in the carlactone pathway of Oryza sativa is not
cleaving 9-cis--carotene, but rather converting 10'-apo--carotenal to carlactones, CCD8

14

still cleaves 9-cis--carotene, but due to competition from a more active cleavage enzyme,
CCD7, the cleavage reaction taking place due to CCD8 is negligible.
Currently in vitro assays are being optimized. In theory, the in vitro assay is expected to
provide more accurate results because of the control over the added concentration of
substrates, whereas in the in vivo assay it is impossible to control. However, the reason it was
easier to obtain results from the in vivo assays in comparison to the in vitro assays during
experimentation is because the bacteria, the host of the reaction, have the optimal conditions
for promoting enzymatic
activity.

On

the

other

hand, in vitro assays serve

as a manmade host for the
reaction
mimic
provided

attempting
the
by

to

conditions
a

living

organism.
Several in vitro assays of
Figure 4.2 Enzymatic activity of CrCCD8.
more than five different
A single enzyme, CCD8, converts 10-apo--carotenal (C27) to Retinal.
substrates have been conducted in order to optimize the experiment. The factors examined for
this optimization include: the substrate concentration and purity, the pH level, the
temperature, and the incubation time (Scopes, 2002). To determine the perfect conditions,
some background about the analogous enzymes and the substrates are considered such as the
light-sensitivity of the substrates and the temperature suitable for enzymatic activity. For
example, a reaction of the four enzymes was done with 10'-apo--carotenal twice with varied
incubation times. The reaction of CrCCD8 with the shorter incubation time produced retinal
(Figure 4.1), while the reaction with shorter incubation time did not. This does not

Hormone Synthesis in Chlamydomonas reinhardtii

15

necessarily deem that longer incubation time is better, because there is a possibility that the
long incubation time allowed for enzyme independent reactions to take place -producing
retinal. While this reaction proved that the enzyme is well folded and that there is enzymatic
activity, the product of the reaction is light sensitive, meaning that it could have isomerized,
yielding only a small amount of retinal for the large amount of 10'-apo--carotenal cleaved.
Modifying the conditions and repeating the in vitro assays must be done to determine the
optimal conditions for the reaction.
Understanding the enzymatic activities taking place in the model organism C. reinhardtii and
its growth regulators expands our knowledge about the organism and its genus. Since it is
almost impossible to allocate a gene responsible for a certain apparatus in larger organisms, it
is necessary to examine genes of smaller organisms like C. reinhardtii. Additionally, biofuel
production heavily depends on algae due to their natural ability to rapidly replicate. A further
understanding of metabolic reactions in algae and their response to growth conditions is
required for biotechnological research and applications. Therefore, allocating hormone
pathways of strigolactones and other similar growth regulators can be deemed necessary to
carry out research in different fields such as synthetic biology and biotechnology.

References

16

Alder, A. et al. (2012). The Path from -Carotene to Carlactone, a Strigolactone-Like Plant
Hormone. Science, 335, 1348-1351.
Sand, Paul F & Eplee, Robert E & Westbrooks, Randy G., 1953- (1990). Witchweed research
and control in the United States. Weed Science Society of America, Champaign, IL
Samarrai, F. (2009, August 27). U.Va. Scientists Identify Gene for Resistance to Parasitic
'Witchweed'. UVA Today. Retrieved June 18, 2014, from
http://news.virginia.edu/node/9543?id=9543
Agricultural Technology Foundation. (2005, June 13). Controlling witChweed in Sub
Saharan Africa. Harnessing the Potential of Public/Private Partnerships, n/a, 8-13.
Davies, P. J. (1987). Plant hormones and their role in plant growth and development.
Dordrecht: M. Nijhoff.
Albabili S., Cabrero A., personal communication, April-June, 2014
Cabrero A., Baz L., personal communication, June, 2014
Smith, S. (2014, March 31). Q&A: What are strigolactones and why are they important to
plants and soil microbes?. BMC Biology. Retrieved July 14, 2014, from
http://www.biomedcentral.com/1741-7007/12/19
Scopes, R. (2002). Enzyme Activity and Assays. In ENCYCLOPEDIA OF LIFE SCIENCES
(Vol. 1, p. n/a). n/a: 2 Macmillan Publishers Ltd. From
http://www.life.illinois.edu/biochem/455/Lab%20exercises/B-gal/enzymology.pdf

Appendix A:
Cloned vectors:
1. D27:

2. CCD7:

Hormone Synthesis in Chlamydomonas reinhardtii

17

Figure A.3 Cloned pThio vector with CCD7 gene of C. re

Figure A.1 Cloned pThio vector with D27 gene of C. reinhardtii.
displaying
theThese
restriction
sites on
both
the
Vector visual displaying the restriction sites on both theVector
vector visual
and the
gene itself.
restriction
sites
are
usv

Figure A.4 CCD7 function in Oryza sativa.

F
CCD7 is the second enzyme in the pathway to carlactones in Oryza sati
D

18

Hormone Synthesis in Chlamydomonas reinhardtii

19

Figure A.5 Cloned pThio vector with CCD8 gene of C. reinhardtii.

Vector visual displaying the restriction sites on both the vector and the gene itself. These restriction sites are used when performing di

Figure A.6 CCD8 function in Oryza sativa.

CCD8
is the
third
enzyme
in the
pathway
to carlactones
in reinhardtii.
Oryza sativa. It oxygena
Figure
A.7
Cloned
pThio
vector
with NCED
gene of C.
Also,Vector
NCEDvisual
is andisplaying
analog to CCD8,
therefore,
and
theory,
they would
be making
t
the restriction
sites
oninboth
the vector
and the
gene itse

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