A functional detection system works like the basic functions of a computer, where you have input, processing and output. Following this comparison for our chemical weapon degradation product detection system P.L.A.N.T., the input is recieved by our engineered receptors, whereas the processing is our signaling cascade inspired by bacterial chemotaxis, which was adapted for plants in 2011 [1]. We intend an output of our detection system to be visible to the naked eye. The leaves of plants are mostly colored green, but plants can also synthesize a large variety of colorful pigments, as visible in many fruits or flowers. We take advantage of this large variety of pigments to establish two different reporter systems for our output. The first pigment is betacyanin (a betalain), which is best known for giving beetroot its intensive red color. For our reporter, we use three enzymes of the tyrosine-derived betalain biosynthesis, which were combined into one open reading frame and recently published as the reporter RUBY [2]. As an alternative to the RUBY reporter, we also developed ANTHOS, a reporter system based on the pigment anthocyanin ANTHOS consists of three transcription factors, which activate the natural anthocyanin biosynthesis in N. benthamiana. We were successfull in transforming the RUBY reporter in our chassis N. benthamiana using transient transformation with Agrobacterium tumefaciens. Under the control of the constitutive CaMV 35S promoter, RUBY colored the leaf bright red. Furthermore, we were able to activate the expression of RUBY with an estradiol inducible expression system, which also resulted in the production of red pigments. We also transiently transformed the ANTHOS reporter in N. benthamiana leaves. Unfortunately, ANTHOS was not yet able to induce the production of red pigments, an issue we could not address due to the lack of time. After successfully establishing RUBY as reporter system, we can now use the reporter as output of our signaling cascade


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Reporter Proteins

Reporter genes are widely used in both basic research and applied synthetic biology. They enable researchers to investigate cellular processes, such as promoter activity or subcellular protein localization studies while being easy to detect [3].

There are several requirements for a reporter system:
its gene needs to be short, enabling an easy expression and less influence on a fused protein
it needs to be easy to detect
the fewer substrates are required, the easier is its application
it needs a large dynamic range
it needs to be stable against degradation

Most current reporter systems are easy to detect, but still rely on the use of technologies like fluorescence microscopy. Therefore, these methods are not suitable for outdoor application in plants. To overcome this problem, we use two alternative reporter systems in our project that are easily detectable with the naked eye. These two systems are both based on natural plant pigments which color the plant red.
Figure 1: biosynthesis pathway of betalain using the three enzymes of the RUBY reporter: CYP76AD1α, DODAα, glycosyl transferase (a). In RUBY, all enzymes are combined in a single open reading frame and separated by 2A peptides (b). Figure was taken from [2].

RUBY Reporter

How Does RUBY Work?

The first reporter system we use in our project is the recently published RUBY, which is based on the plant pigments betalains [2][4]. They belong to one of the major pigment classes in plants, are water-soluble and have a strong antioxidant activity [5]. Betalains can be divided into two groups, the yellow betaxanthins and the red-violet betacyanins with absorption maxima at 460-480 nm and 535-538 nm, respectively [4]. The final products of the RUBY reporter are red betacyanins.

Although the betalain biosynthesis is naturally based on a complex network of enzymes and intermediates, RUBY uses only three enzymes to convert tyrosine into betalain: a cytochrome P450 (encoded by CYP76AD1α), DODAα and a glycosyl transferase. These enzymes are encoded in a single open reading frame. They are separated by 2A peptides, enabling self-cleavage of the fusion protein to obtain three individual and functional enzymes (see figure 1).

RUBY: Proof of Concept

To assess the properties of the RUBY reporter, we transiently transformed RUBY under control of the constitutive CaMV 35S promoter in leaves of N. benthamiana plants. Upon transformation with 35S:RUBY, the leaf color changed clearly visible to red during the following days (see figure 2). Even at the day of WikiFreeze (21st October 2021), the leaves were still showing a vibrant red coloration, confirming that the RUBY pigments stay stable in plant leaves for a long time (Fig. 3). Several kinds of analyses of this color change were performed, which are presented in the following paragraphs.

Figure 2: N. benthamiana leaves infiltrated with 35S:RUBY, from 1 to 8 days after infiltration with agrobacteria for transient transformation.
Figure 3: N. benthamiana leaves infiltrated with 35S:RUBY, from 1 to 9 weeks after infiltration with agrobacteria for transient transformation.

Betalain Extraction & Quantification

Betalains are the final products of the RUBY reporter. To investigate how the betalain content of transformed leaves changes over time, we extracted and quantified betalain 1 day, 2 days, 3 days, 7 days, 8 weeks and 9 weeks after transformation. For extraction, 1 cm² of the leaf was cut out and weighed. A small amount of extraction buffer (35 % EtOH, pH 5) was added to the sample, which was then lysed with a ribolyzer (Precellys 24). After the lyses, the volume of the extraction buffer was added up to 1 mL. After centrifugation, the supernatant was transferred in cuvettes and the absorption at 535 nm was measured in a spectrophotometer. In the end, the betalain content in mg per g fresh weight was calculated with the following formula:

BC(mg/g) = [A*(DF)*(MW)*Vd/ε*L*W]

A: absorption value at the absorption maxima of 535 nm for betacyanins
DF: dilution factor
MW: molecular weight
Vd: solution volume [mL]
ε: extinction coefficient of betanin (60.000 L/(mol cm))
L: path length of cuvette [cm]
W: fresh weight [g]

The analysis shows that at the first day after transformation, the betalain content was significantly lower than at all the other time points. At every following time point, the betalain content was high, with a slight decrease from 8 weeks to 9 weeks. This shows that after the transformation of RUBY under the control of the constitutive CaMV 35S promoter, the pigments produced by the RUBY reporter are present in the leaves for a very long time.

Figure 4: N. benthamiana Betalain content of transformed N. benthamiana leaves in mg per g fresh weight at 6 timepoints after agroinfiltration. Timepoints with the same letters (a, b, c) show no significant differences, timepoints with different letters have significant differences. Statistical test was the t-test.
Figure 5: Relative Expression of RUBY genes (CYP76AD1α, DODAα, glycosyl transferase) at different time points (day 2 and day 7). RT-qPCR was performed with N. benthamiana leaves that transiently express RUBY after agroinfiltration. Data was normalized to actin and plotted against highest expression level. Statistics were performed with unpaired t-test (***, p < 0,001; **, p < 0,01; ns, not significant).

Transcript Analysis

To ensure that all three RUBY enzymes were expressed, we performed a transcript analysis at two different time points after agroinfiltration to analyze the relative expression of CYP76AD1α, DODAα and glycosyl transferase. The time points day two and day seven were chosen, because transient expression with agroinfiltration is highest after two days and should decrease over time [6].

The results show that all three genes responsible for the betalain production are in fact expressed after two and seven days. Furthermore, we were also able to show that the expression is significantly higher at two days in comparison to seven days for the genes CYP76AD1α and DODAα (Fig. 5).

Metabolome Analysis

Betalains occur exclusively in a few families of the core Caryophyllales [7]. In every family, except for Simmondsiaceae, Macarthuriaceae, Caryophyllaceae, Limeaceae, Kewaceae and Molluginaceae, betalains are produced instead of anthocyanins or the pigment state is unknown [8]. Our model organism Nicotiana benthamiana, which we are using for the experiments, therefore does not possess endogenous betalain biosynthesis. Therefore, we discussed possible consequences with Prof. Dr. Boas Pucker, expert on plant genomics and specialized plant metabolites, who hypothesized that the expression of RUBY might lead to a metabolic imbalance, due to a possible tyrosine drain when used as precursor for RUBY. To investigate this hypothesis in detail, we used GC/MS (gas chromatography in combination with mass spectroscopy) to analyze the levels of a variety of different metabolites. These analyses were performed with untreated N. benthamiana, leaves und compared to the ones with RUBY expression. Therefore, N. benthamiana leaves were transiently transformed with RUBY, under the control of the constitutive CaMV 35S promoter, using Agrobacterium tumefaciens. A detailed description of the results of the metabolome analysis can be found here.
Figure 6: Photosystem II yield at normal light conditions compared between not transformed control areas and with RUBY transformed areas of N. benthamiana leaves.
Figure 7: Light curve of Photosystem II yield of a dark adapted N. benthamiana leaf compared between not transformed control areas and with RUBY transformed areas.
Figure 8:Light curve of non-photochemical quenching (NPQ) of a dark adapted N. benthamiana leaf compared between not transformed control areas and with RUBY transformed areas.

PAM Measurement

The measurement of chlorophyll fluorescence is a powerful tool to investigate and elucidate the complex regulation of photosynthesis. This is due to the tight connection of the three possible reactions of de-excitation of the chlorophylls in photosystem II (PSII): photochemical reactions, emission of fluorescence and emission of heat. The portable chlorophyll fluorometer “MINI-PAM”, built by the Heinrich Walz GmbH, is a great device to record and evaluate different characteristics of chlorophyll fluorescence. The most prominent are the yield of photosystem II and the non-photochemical quenching (NPQ), the process of enhanced conversion of absorbed light energy for harmlessly dissipating excess excitation energy. The PAM measurement is a very complex process. But in the end, the information is assessed by two consecutive measures of fluorescence yield, one briefly before and one during a short pulse of saturating light.

To assess the influence of the expression of the RUBY reporter on the efficiency of photosynthesis, we performed two different experiments with the MINI-PAM to measure three different parameters. As first experiment we measured the PSII yield at ambient light conditions of infiltrated and control areas of each two leaves of in total twelve plants, with three replicates of each measurement. In the second experiment, we measured the light curve of a dark adapted leaf with five replicates for the infiltrated area and the control area. With this light curve, it was possible to measure both PSII yield and NPQ depending on the light intensity.

The measurement of PSII yield at ambient light conditions in the first experiment shows that the leaves in both conditions, expressing RUBY and control, both show the same PSII yield of a bit less than 80 % (see figure 6). This indicates that the expression of the RUBY reporter does not impair the photosynthesis at normal light conditions.

The light curve experiment shows that with increasing light intensity the PSII yield of the leaves expressing the RUBY reporter is slightly decreased compared to not transformed areas of the leaves (see figure 7). Apparently, the expression of the RUBY reporter impairs the ability of the leaf to cope with high light intensities. At the same time, the expression of RUBY leads to increased NPQ with increasing light intensity (see figure 8).

Figure 9: N. benthamiana leaves three days after transformation with 35S:RUBY (A) and lexA:RUBY (B)

Estrogen Induced Expression of RUBY in Nicotiana benthamiana

In order to find out if RUBY is not only expressible with a constitutive promoter, but also with an inducible one, we used an estrogen-based induction system. There, RUBY was put under the control of the lexA promoter (BBa_K3900024). A slight red coloration of the leaves was visible. Consequently, RUBY is suitable as an inducible reporter in plants. This can also be confirmed by recent research of Alfonso Timoneda et al., who used betalain pigments to visualize arbuscular mycorrhizal colonisation [9].

Anthocyanin Reporter

Wild type Nicotiana benthamiana is not capable of betalain synthesis. This prevents false-positive results when the RUBY reporter is not activated. However, the induction of betalain synthesis might cause a harmful metabolic imbalance. To prevent this, we decided to develop an additional reporter system based on anthocyanins. These pigments occur naturally in N. benthamiana leaves. Therefore the metabolism should be better adapted to cope with anthocyanin biosynthesis. We call this newly developed reporter system ANTHOS.

Anthocyanin Biosynthesis

As explained above, anthocyanins are, like betalains, one of the most common plant pigment groups and belong to the flavonoids, a large class of specialized metabolites in plants. Anthocyanins are responsible for the red, purple and blue colors of many flowers and fruits and attract pollinators and seed dispersers [10]. The synthesis of anthocyanins plays a role in the plant’s protection from light and pathogens and is induced under stress conditions. The regulation of anthocyanin biosynthesis is highly conserved in angiosperms. To activate the anthocyanin production, a so-called MBW complex is required. It consists of a R2R3MYB transcription factor, a bHLH transcription factor and a WD Repeat Protein (WDR) [11]. The bHLH transcription factor and the WDR protein fulfill several functions beyond anthocyanin regulation, while the involved R2R3-MYB transcription factor specifically regulates the production of anthocyanins.

To identify which combination of transcription factors we should express for a functional anthocyanins-based reporter system (ANTHOS), we talked to Dr. Ralf Stracke, expert on transcription factors at Bielefeld University. In fact, he annotated the R2R3-MYB transcription factor family in 2001 [12]. He recommended using the proteins AtMYB75, AtbHLH2 and AtTTG1 of Arabidopsis thaliana.

For the cloning of ANTHOS, we used two different approaches. For the first approach, we cloned each of the three transcription factors in one separate open reading frame with its own promoter and terminator. The promoter for each of the three genes is the estrogen inducible lexA promoter. As terminators, the G7 terminator was used for AtMYB75 and AtbHLH2, the TUB9 terminator for AtTTG1. For the second approach, we used one open reading frame for the expression of the transcription factors, separated by 2A peptides, which are also used for the RUBY expression. Like in the first approach, we chose the lexA promoter combined with the G7 terminator. We decided to use these combination of promoter and terminators based on a recent publication from Andreas Andreou and Nayomi Nakayama, where they investigated promoter-terminator interactions. We used terminators, which showed a high expression in combination with the lexA promoter.

Andreas Andreou also developed the cloning method Mobius Assembly in Nayomi Nakayama’s lab, which we used for cloning ANTHOS. This method is based on Golden Gate cloning systems, while the backbones are binary plasmids. These plasmids are suitable for plant transformation. In Mobius Assembly, basic parts like promoters, terminators and coding sequences are first cloned into level 0 vectors. In level 1, several basic parts are combined to form a transcriptional unit (TU). In level 2, TUs are assembled to form a multi-TU construct.

Estrogen Induced Expression of Anthocyanin in Nicotiana benthamiana

To establish our anthocyanin based reporter system ANTHOS, we transiently transformed our level 2 vectors, containing both the estrogen responsive transcription factor and the transcription factors of ANTHOS, into N. benthamiana leaves. One day after transformation, estrogen was applied to the leaf. For this, several methods were tested: applying it with a brush, injecting it directly into the leaf and applying a lanolin paste containing estrogen. Unfortunately none of the treated leaves showed a change in leaf color.


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