An analysis of the presence of proteins on different levels in dark grown and light grown plants

Biogenesis of organells

Project Report 2014 32 Pages

Biology - Botany


Table of contents

1. Introduction

2. Experiments on biogenesis of plastids
2.1. Part A: Transcript level
2.1.1. Material and Methods RNA isolation from Arabidopsis thaliana Photometrical analysis of RNA concentration RNA gel cDNA synthesis Test PCR Quantitative real time PCR Quantification of real time PCR
2.1.2. Results Photometrical analysis of RNA concentration RNA gel Quality control of real time PCR amplificons Quantification of real time PCR
2.1.3. Discussion on transcriptional level on biogenesis of plastids
2.2. Part B: Protein level
2.2.1. Material and Methods Protein extraction Determination of protein concentration (Bradford reagence) Coomassie gel Western Blot and Antibody stain Isolation of plastids in Organello labelling
2.2.2. Results Protein concentration by Bradford Coomassie gel Western Blot with Antibody stain SDS-Page of in Organello labelling by isolation of plastids
2.2.3 Discussion
2.3. Part C: Light-harvesting complexes
2.3.1. Material and Methods Isolation of thylakoid membranes from Arabidopsis thaliana seedlings Chlorophyll isolation and pigment quantification
2.3.2. Results Semi-native polyacrylamide gel Chlorophyll quantification
2.3.3. Discussion

3. Discussion of the experiments on biogenesis of plastids


1. Introduction

Chloroplasts are the essential organelles for photosynthesis, found in land plants and photosynthetic organisms. The development of chloroplasts is a complex pathway with the participation of several different proteins. The source of all plastids is the proplastid, which can develop to Chloroplast, Etioplast, Chromoplast and other kinds of plastids, depending on the environmental conditions (Fig. 1).

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Figure 1: Development of plastids out of proplastid; (source: www.spektrum.de)

If the plant is kept in dark, the proplastids develop to etioplasts, which are not photosynthetically active and contain the prolamellar bodies (Fig. 2a). The investigations of etioplasts and prolamellar bodies showed the presence of proteins, which are important for photosynthesis, for example the Cytochrome b6f complex (Kanervo et al., 2008). There are also proteins, which are mainly found in etioplasts including FtsH protease complex, which is responsible for biogenesis of multiprotein complexes, and the POR proteins (Kanervo et al., 2008). POR proteins are encoded in the nucleus and are imported into the chloroplast as transit peptides (Kim and Apel, 2004). There are three isoforms of POR proteins: PORA, which is only found in etioplasts and degraded under light exposure, PORB and PORC, which are both detected in seedlings and older plants. The POR proteins have an important function in the biosynthesis of chlorophyll, where they catalyse the reduction of protochlorophyllide (Phlide) to chlorophyllide (Chlide) under light influence (Kim and Apel, 2004).

Etioplasts are completely free of pigment complexes, Photosystem II, Photosystem I and LHC complexes (Kanervo et al., 2008). Under light exposure, etioplasts are transformed into chloroplasts, which causes the dispersion of the prolamellar bodies and changes in the protein levels (Fig. 2b).

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Figure 2: a) Prolamellar body (PLB) in etioplast. b) PLB after one minute of light exposure.

(Source: www.biologie.uni-hamburg.de)

Photosystem complexes are being formed and the chlorophyll is synthesized, in order to enable photosynthesis. An increase of the protein D1 is detected, which is essential for the Photosystem II assembly and function. The AtCtpA gene encodes the D1 protein, which is translated during de-etoilation and inserted into thylakoid membrane (Che et al., 2013). Furthermore photosystem I, photosystem II, Cytochrome b6f complex and ATP synthase are built. The transmembrane Alb3 and Alb4 are responsible for the organisation of the assembly of those complexes. Further investigation showed that alb4 mutants have retarded growth, smaller cells, less chloroplasts, altered thylakoid structures and decrease in levels of ATPase subunits (Benz et al., 2009). This proves the importance of Alb4 in relation to the stability of the ATPase complex. Alb3, a homologue to Alb4, which is mostly located in the thylakoid membrane, is responsible for the assembly of chloroplast enzyme complexes and membrane biogenesis (Sundberg et al., 1997). Furthermore an increase of dsbA protein is detected, which is a thioloxidoreductase and important for the assembly of the photosystem II complex. The dsbA mutants show impaired plant growth, PSII deficiency and accumulation, while PSI is not affected (Karamoko et al., 2011). Another important step in the biogenesis of the chloroplast is the formation of light-harvesting complexes (LHC), which absorb light and transfer the energy to PSI and PSII. The LHC are formed by the transmembrane located light harvesting chlorophyll-binding proteins (LHCP), while LHCA proteins assemble the LHCI complex and LHCB affects the formation of LHCII complex.

LHCI is connected with PSI, while LHCII is a subunit of PSII (Andersson et al., 2003). Analysis of lhcb1 and lhcb2 mutants showed a severely impact on the phenotype due to lack of the LHCP genes. Those plants had a pale green appearance and reduced chlorophyll content (Andersson et al., 2003). The light-harvesting complexes are connected to chromophores, which allow the absorption of light. We differentiate between carotenoids, chlorophyll a and chlorophyll b, which absorb light at different wavelengths. Both pigments are not found in etioplasts and develop during the biogenesis of chloroplasts like most of the other proteins and complexes, which are related to photosynthesis.

In our experiments we want to analyse the presence of those proteins on different levels, comparing dark grown and light grown plants.

In part A the samples are being analysed on transcript level, determining the amount of transcripts for LHC, POR and ALB4. Here we also compare transcription levels of those proteins in Wild-type plants and Δ dsbA mutants, which have PSII deficiency (Karamoko et al., 2011). As a control the 18S rRNA is being used, which is a housekeeping gene and should be equally expressed in all samples.

In part B we will compare the same samples on protein level by protein isolation, Coomassie staining and immunostaining with antibodies. Here we also focus on the proteins ALB4, LHC and POR. Furthermore, in organelle labelling was performed, which should show differences in protein levels between chloroplasts and etioplasts.

Part C is focusing on the light-harvesting complexes, which are developed during de-etoilation of dark grown seedlings. A semi-native gel electrophoresis was performed and chlorophyll a and chlorophyll b amounts were measured. As samples the wild-type, Δ dsbA and alb3 mutants were used and compared to each other.

2. Experiments on biogenesis of plastids

2.1. Part A: Transcript level

2.1.1. Material and Methods RNA isolation from Arabidopsis thaliana

The four samples (WT light grown, WT dark grown, Δ dsbA mutant light grown, Δ dsbA mutant dark grown) were grinded with mortar and by adding liquid nitrogen to fine powder. Isolation buffer (β-mercaptoethanol 1:100) was added and the samples vortexed and incubated at room temperature. The samples were transferred on QIAshredder columns and centrifugated. The supernatant was transferred to a new Eppendorf tube and 0.5 volume parts of EtOH was added. After mixing solutions, the samples were added on RNeasy columns, centrifugated and the flow-through was discarded. RW1 buffer was added and the column centrifugated.
10 µl DNase I was mixed with RDD-buffer and added to the column. After incubation, the column was washed with RW1 buffer. The flow-through was discarded and the column washed with RPE-buffer twice. After discarding the flow-through, the residual EtOH was dried out by centrifugation. In order to elute the RNA, 50 µl RNase free water was added on the column and centrifugated. The isolated RNA was freezed at -20°C. Photometrical analysis of RNA concentration

The RNA quantity was analysed photometrically, measuring the absorptions of the nucleic acids at a wavelength of 260 nm, 280 nm and 320 nm. The ratio of 260/280 nm serves to determine protein contaminations. The measurement at 320 nm is used as a control for background correction, since nucleic acid and proteins do not absorb at this wavelength. The concentrations of the RNA samples were calculated with a given formula.

Abbildung in dieser Leseprobe nicht enthalten (1) RNA gel

Furthermore, the samples were loaded on a RNA-Gel in order to determine the quality of the isolated RNA. The RNA gel was produced with agarose, 10x MOPS buffer, formaldehyde and EtBr. The samples were denaturated and loaded onto the gel (1.5 µg). 1x MOPS buffer was used as running buffer and the RNA was separated for 2 hours. The result was photographed under UV-light. cDNA synthesis

Since the RNA is not stable for PCR and usually gets denaturated after a few cycles, cDNA was prepared by reverse transcriptase, using the BioRad iScript cDNA Kit. Due to low RNA concentrations, only 0.6 µg RNA (see script: 1 µg) was mixed with 5x buffer and 1µl reverse transcriptase for each sample. Those dilutions for the realtime PCR were incubated in a PCR machine with programmed incubation times at 25°C, 42°C and 85°C. Test PCR

The cDNA quality was first determined for all samples, focusing and amplifying the 18S rRNA gen, which is a “housekeeping gene” and therefore always stable at any environmental conditions. A Master Mix was prepared for all samples, with primer dilutions of 1:100, dNTP (20mM), MgCl (25mM), buffer (10x), Taq Polymerase (5U/µl) and 1µl cDNA. Furthermore, a negative control with water instead of cDNA was prepared. The samples were incubated in a thermocycler with 30 programmed cycles. The amplification products are analysed by agarose gel electrophoresis, using 1.5% gel, 1x TAE running buffer and EtBr. 8 µl of the PCR samples were mixed with 2 µl Loading buffer (1x), loaded onto the gel and the electrophoresis was performed at 100V for 20 minutes. Quantitative real time PCR

The real time PCR is performed with a fluorescent dye (SyberGreen), which is detected for every cycle and amplification product and therefore proportional increasing with accumulation of double stranded amplicons.

The Master Mix for the real time PCR was prepared, using primer for 18S gene and POR gene (1:20 dilution), cybergreen (BioRad) and water. The Master Mix was pipetted into a
96-well microtiter plate and the cDNA samples (1:10 dilution) were added. The plate was placed in a MyiQ cycler (BioRad) with 50 programmed PCR cycles.

Furthermore, a quality control of the real time PCR amplicons was performed, using 1.5% agarose gel. Quantification of real time PCR

The expression levels of POR, ALB4 and LHC genes were compared to expression of the reference gene 18S, using the given Pfaffl-formula (Fig. 5).

2.1.2. Results Photometrical analysis of RNA concentration

The extinction of all RNA samples was measured at 260, 280 and 320 nm. The samples were diluted to a dilution factor of x50. The 260/280 ratio shows the pureness of the RNA solution, while pure RNA should have a ratio of 1.8 – 2.0. The concentration was calculated according to the law of Lambert Beer:

Abbildung in dieser Leseprobe nicht enthalten (2)

The results from the measurement were combined into a table:

Table 1: Measured RNA extinctions and calculated RNA concentration per Lambert Beer law.

illustration not visible in this excerpt RNA gel

For the determination of RNA quality, the RNA gel was photographed under UV light (Fig 3). We have 4 RNA samples: wild-type light, wild-type dark, and mutant light and mutant dark. In the lane for WT light, WT dark and mutant dark are no bands visible. Only mutant light lane has a smear of bands and one single band at the bottom. On the left side are the results from another group.

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Figure 3: RNA gel; Results at the right side (WT=wild-type, M = mutant, L = light, D = dark). Quality control of real time PCR amplificons

After the real time PCR, the quality of the amplification products was determined on agarose gel and furthermore the result photographed under UV light (Fig. 4). Our samples are located at the left; one band is visible below the 500 bp mark of the marker for every sample.

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Figure 4: Quality control of real time PCR products. First lane: λ pst standard, right side: results from other groups. Quantification of real time PCR

In order to calculate the expression levels of the three protein genes, we were provided with Ctvalues and PCR efficiency values by the MyiQ cycler. We received two values per protein, since always two groups were working with one certain protein primer pair. Therefore, the mean value of two samples was used for the calculations. Every group has their own values for the 18S mRNA gene, which we will refer to in our calculations. The given Pfaffl formula for gene expression:

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Figure 5: Pfaffl formula for calculating relative expression ratio R.

(Source: www.gene-quantification.de)

The given Ct- and PCR efficiency values were combined in an Excel-file (Table 2). The power values were calculated with the Pfaffl formula. Our groups’ Ct values for the POR gene were not presentable, and got therefore replaced by real time PCR values from last years practical course group.

Table 2: Ct values and PCR efficiency for 18S mRNA (control gene), POR, LHC and ALB4. PCR eff. = PCR efficiency. Mean eff. = mean efficiency.

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Table 3: ratio of power values, referred to 18S mRNA gene values.

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The relative expression ratios were illustrated in graphs, comparing the expression levels of each gene from wild-type light/dark and mutant light/dark samples (Fig. 6 – 9).

Figure 6: Relative expression ratio for PORA gene.

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Figure 7: Relative expression ratio of PORB gene.

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Figure 8: Relative expression ratio of PORC gene.

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Figure 9: Relative expression ratio for LHC gene.

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Figure 10: Relative expression ratio for ALB4 gene.

2.1.3. Discussion on transcriptional level on biogenesis of plastids

The RNA gel in Figure 3 shows no bands for the samples wild-type light, wild-type dark and ΔdsbA mutant dark. This could mean that the RNA isolation wasn’t successful and therefore no RNA was in our samples. But the photometrical analysis shows, that there is RNA in all samples, even though wild-type light and wild-type dark has a very small amount. Therefore, the loaded amount of RNA was not enough and could not be detected by the UV light. The wild-type dark sample has also some protein contamination, indicated by the high value for 260/280nm ratio. The inconsistency between measured RNA concentrations and the absence of bands on the RNA gel could mean that the RNA isolation was not properly performed or mistakes during the photometric analysis happened. The smear in the third lane, for sample mutant light, could indicate protein contamination and also inaccurate RNA isolation, which again contradicts the 1.86 value for the 260/280nm ratio, which indicates a pure RNA solution. The RNA samples on the left on figure 3 shows, how the result should look like. Several RNA separation products are clearly visible. The different expression level of bands between lanes 1-2 and lane 3-4 indicates here also, that different amounts of RNA were loaded.

The quality control of the real time PCR amplicons shows that the real time PCR was successful and resulted in PCR products around 300 bp (Fig. 4). The accurate length of the product is difficult to determine, because it is out of range of the marker. The expression level of the bands is different for each lane. Wild-type light has the weakest band, which indicates that there is less POR gene expression in wild-type light grown plants.

Figure 7 shows the expression level of the PORB gene, comparing all four samples, while wild-type light serves with a value of 1 as a reference for the other samples. It is clearly visible, that wild-type dark grown samples have a much higher expression of the POR gene with a value of around 4.4. This result correlates with our expectation, that POR is located mainly in the prolamellar bodies in etioplasts and degraded during de-etiolation through catalysis of protochlorophyllide to chlorophyllide. Therefore we have less gene expression of PORB in light-grown plants. The mutant light-grown plants have a PORB gene expression value of 0.7, which could indicate, that the dsbA gene is not only important for PSII function, but also could have an influence on the PORB gene expression. The dark grown mutant has a PORB expression value of 3.2, which also correlates with the assumption, that dsbA mutants have an impaired gene expression of POR. But as expected, the value is much higher than in the light-grown mutant, because the PORB is mainly located in the etioplasts before de-etiolation.

Figure 6 compares the expression level of PORA of the four samples.

With a value of 111.9 and 112.8, the dark grown samples have an extremely stronger expression of PORA compared to the light grown samples. This shows that PORA is degraded completely after de-etiolation and can be only found in etioplasts.

In contrast to that, PORC has the higher values for the wild-type light grown and mutant light grown (Fig. 8). It seems that the expression of PORC is impaired by the mutation, since the light grown mutant has only a value of 0.4. The result shows that the PORC gene is expressed more after de-etiolation, in contrast to the PORA and PORB genes.

Figure 9 illustrates and compares the expression level of the LHC gene between the four samples. Wild-type light serves here again as a reference with a value of 1. The sample wild-type dark has a much lower value of 0.14, showing that there is almost no expression of LHC gene in the dark. This represents our expectations, since the light-harvesting complexes are only formed during de-etiolation, together with production of pigments. The plant doesn’t need the LHC in the dark, since no light is available to get absorbed and therefore no light-harvesting complexes are being formed. The mutant light-grown sample has an expression value of 0.7 and therefore lower LHC gene expression than the wild-type. This shows that not only POR could be affected by the mutation, but also the expression of LHC is impaired in dsbA mutants. Most probably the LHCB gene expression is affected, since LHCB is connected with LHCII complexes and therefore connected to the PSII, which is deficient in dsbA mutants. The mutant dark-grown sample has an expected gene expression value of 0.1, which indicates the very low LHC expression in etioplasts.

The expression level of the ALB4 gene is represented in Figure 10. Compared to the wild-type light grown sample, the dark-grown sample has a value of only 0.28. This shows that the ALB4, which is responsible for the assembly of the ATPase complex, is mostly translated after de-etiolation, since the photosystems are not required during etiolation. The ALB4 expression value for mutant light-grown is 0.55, so the mutation at the dsbA gene has also a negative influence on the ALB4 expression. Also for the mutant, the dark-grown sample has a lower ALB4 gene expression level with a value of 0.29.

All three examples of gene expression indicate that the dsbA mutation seems to have more impact on other genes than expected. DsbA mutants not only have a PSII deficiency, but are also impaired in expression of POR, LHC and ALB4 genes, which are important for pigment synthesis, light-harvesting complexes assembly and building ATPase complexes.



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Biogensis Organelles Transcription Protein Light-harvesting complex Arabidopsis thaliana Chlorophyll qPCR




Title: An analysis of the presence of proteins on different levels in dark grown and light grown plants