In higher plants, phototropin is in charge of controlling the movements such as phototropism, chloroplast relocation and stomatal opening so that the plant can make the best use of sun light while avoiding the harmful effect which may be caused by too strong sun light (Briggs and Christie, 2002). As a single-celled organism, C. reinhardtii exhibits phototactic behavior. It has been suggested to be triggered by microbial-type rhodopsin (Foster et al., 1984; Lawson et al., 1991; Takahashi et al., 1991). It seems that the blue light receptor phototropin has little effect in helping cell to select the best place for growth. And till now, phototropin has only been reported to be involved in the sexual life of C.reinhardtii (Huang et al., 2002; Huang et al., 2003).

In Huang et al., (2002), quantitative competitive RT-PCR and western blot were applied to determine the level of phototropin in different growing stages. In that experiment, vegetative cells, cells grown in darkness, gametes directly generated from vegetative cells, pregametes, and gametes generated form pregametes had been tested. Among those different kinds of cells, both the phototropin level and phototropin mRNA level were the lowest in vegetative cells, but phototropin levels in other cell types are similar (Huang et al., 2002). Till now the function of phototropin in vegetative cells has not been reported. Is phototropin unimportant in vegetative cells? The answer is no. In this thesis, vegetative cells of different strains grown under same light conditions were tested. The levels of phototropin in these cells were different (Figure 3.1.1.2 A). Then vegetative cells from three different strains grown under same conditions were fractionated. It was discovered that the phototropin distribution also varied in these cells (Figure 3.1.1.2 B). The level of phototropin in vegetative cells is very delicately regulated, which suggest that slight change in its level may bring harmful damage to the cell. Vegetative cells grown in light or darkness were fractionated. The distribution and level of phototropin was different under these two conditions (Figure 3.1.2.1 D). Light is one factor that is in charge of regulating phototropin level and distribution. But obviously, light is not the only factor. The other factors are to be discovered.

For the first time, it was shown that phototropin existed as a soluble protein in Chlamydomonas. It was reported to be an insoluble protein in Chlamydomonas in Huang et al., (2002), Huang et al., (2004). It was shown in this thesis that no matter in exponential stage of vegetative cells, stationary stage of vegetative cells or in gametes, phototropin existed as an soluble protein as well as a membrane attached protein (Figure 3.1.2.1) Although the soluble fraction only counts for small amount of total soluble protein, it opened a chance for purification. From the amino acid sequence analysis, it is known that phototropin has no hydrophobic region which allows integration into the membrane. In Chlamydomonas, phototropin could attach to the membrane by some adapter protein or by binding to a second messenger such as DAG (diacylglycerol). Or, it could be covalently modified by a fatty acid. Some detergents and chaotropic salts have been tried to wash it off the membrane, but the efficiency was very low (data not shown). This suggests that other methods are required to solubilize the protein.

Figure 4.1.1 Phosphorylation prediction of C. r. phototropin (NetPhos 2.0 Server).

According to the prediction, there are 20 Ser, 10 Thr and 4 Tyr phosphorylated in phototropin after excitation. The phosphorylation will increase the molecular weight ~2.7 kD.

RNAi technology is a popular tool to silence target genes, however, the enzymes which attend the process have not yet been identified in Chlamydomonas. The reason to use RNAi technology to reduce the phototropin level in Chlamydomonas lies in the fact that it is not yet practical to knock out of Chlamydomonas endogenous gene by homologous recombination. Recently, homologous recombination carried out by single strained DNA shed the light on the possibility of using homologous recombination to knockout endogenous gene in Chlamydomonas (Zorin et al., 2004). However, trials to knockout phototropin gene in Chlamydomans by this method were unsuccessful in this work.

Fuhrmann et al., (2001) put forward the hypothesis that Chlamydomonas had the RNAi mechanism and also proved the assumption by silencing a Chlamyopsin gene (cop). The best construct for silencing shown in that paper was composed of a leading genomic fragment fused to a reverted cDNA counterpart under the control of the cop promoter. The authors believed that after transcription and RNA splicing, a perfect hairpin structure would form and trigger the RNAi process. The function of those introns in the leading genomic piece was to prevent recombination potential of the hairpin structure during cloning in E.coli and to assist in maintaining the expression level of transgene similar to thatof the authentic cop gene. cw15 arg- A was used as recipient strains. It seems that in cw15 arg- A, the RNAi construct works perfectly. Half of the transformants obtained by co-transformation with marker plasmid pSI103 showed a significant reduction in their opsin content (Fuhrmann et al., 2001).

A similar strategy was applied to silence the phototropin gene in this thesis. An artificial genomic fragment was used in place of the authentic genomic fragment of phototropin. The construct seemed to work well in strain cw15 arg- A. 12.5% of the transformants showed a reduction of phototropin of around 95%. However, this RNAi construct did not work properly in other strains. Although intensive transformation and screening had been carried out, only one clone with a less pronounced reduction level of phototropin was found. It seems that different strains may have different sensitivities to the same RNAi construct, which could be caused by the integration position of the construct. Since the selection marker and RNAi construct were in different plasmids, co-transformation efficiency may also affect the number of positive clones.

The efficiency of RNAi silencing was different under different growth condition. It was found for the transformant C4, the best silencing conditions was low light conditions (Chapter 3.3). According to the mechanism of RNAi, the reduction level should be quite stable under all kinds of conditions since RNAi is a self-amplifying process. The different promoter used instead of the original phototropin promoter could also be an explanation for the phenomenon. The combination of phototropin promoter with piece of phototropin genomic fragment could help to adjust RNAi construct transcripts level more close to phototropin mRNA, i.e., the levels of the two newly synthesized mRNAs could be almost synchronized. Successful silencing has been achieved in this way (Fuhrmann et al., 2001, Huang et al., 2003).

Whether or not to use 3' UTR in the RNAi construct is a interesting problem. The 3' UTR is important to a mRNA, it decides not only the ending of transcription and the tailing but also the delivery, stability and localization of mature mRNA. A successful example has shown that it was not necessary to have a 3' UTR in the RNAi construct for the Chlamyopsin gene (Fuhrmann et al., 2001). As in the formal result obtained with strain cw15 arg- A, the RNAi construct used in this thesis also worked nicely (Figure 3.2.2.1). But in other strains, the strategy seems not so efficient, which suggests that lacking of 3' UTR could be the reason. Several studies already showed that RNAi process was restricted to the cytoplasm (Zeng and Cullen, 2002; Kawassaki and Taira et al., 2003). Lacking 3' UTR could also cause low efficiency in ending of transcription and delivering mRNA out of the nucleus. Since piece of foreign DNA is randomly integrated into C. reinhardtii genome (Kindle et al., 1989), the only identified CC32pab1mt(+) transformant could result from the case that the RNAi construct happened to locate in front of a 3' UTR of one Chlamydomonas gene. Without 3' UTR, tailing of mRNA could be less efficient. Normally, mRNA without polyA tail will enter deadenylation-dependent pathways. It would be degraded either by 3' to 5' exonucleolytic decay or 5' to 3' exonucleolytic decay. In either way, siRNA would not be created. Thus the hairpin RNA would not work properly.

The use of artificial genomic DNA instead of authentic genomic DNA could be another explanation for low silencing efficiency. There are two known RNAi interference pathways. One is the short interfering RNA (siRNA) pathway which starts with double-stranded RNA and form siRNA by Dicer. The other pathway is through miRNA (micro interfering RNA) (Pasquineli et al., 2002). When there is imperfect hairpin RNA structure, Dicer could also digest them into short RNA pieces (miRNA). Those miRNAs are believed to bind to sites that have partial sequence complementarity in the target mRNA. miRNA can direct the cleavage of a perfectly complementary target RNA. When miRNA binds partially complementary target RNA, it blocks the translation (Cerutti, 2003). In the experiment described in this thesis, the artificial genomic piece of phototropin was composed of several small pieces and conjugated with different restriction enzyme sites. After post-transcriptional modification, the two introns would be removed but restriction enzyme sites would still remain, which caused the product to be an imperfect hairpin RNA. After cleavage, RISC guided by small pieces of RNA which would bind to target RNA with few unpaired base would not cleave the target RNA. Thus the self-amplification of RNAi is limited. The different performance of same RNAi construct in different strains could be caused by many reasons. To obtain more phototropin silencing strains, new RNAi constructs need to be made.

In the obtained RNAi construct transformants, both full length phototropin level and truncated version of phototropin level got reduced. The fact further proved that those two proteins originated from one gene. In case that these two proteins carry out different function, it would be interesting and challenging to study the differences.

In Chlamydomonas, blue light was found to be closely related with the sexual life (Weissig, 1991). In Chlamydomonas gametogenesis, the first condition required is nitrogen starvation. When vegetative cells incubated in darkness are deprived of nitrogen in the medium, they develop into pregametes. The second signal is blue light, after which pregametes acquire mating competence and change into gametes. Blue light is also necessary for maintaining the mating competence (Huang and Beck, 2003). Since the relationship between phototropin and gametogenesis has been clearly demonstrated in Huang and Beck, 2003, in this thesis, the focus was placed on the involvement of phototropin in zygote germination.

As mentioned in Gloeckner and Beck, (1995), light is required to trigger meiosis and germination of C. reinhardtii zygotes. Huang and Beck (2003) further put forward the possibility of phototropin involvement in this process. Reduction of the zygote germination rate was found in the offspring of phot- strain (Huang and Beck, 2003). However, the experiment protocol used by the authors of the two papers mentioned above is complicated and difficult to handle. This difficulty limited the usage of that method. The complexity of the process increased the possibility of man-made mistakes. The distinction between vegetative, unmated gametes and zygotes is not absolutely accurate. To overcome these drawbacks, a new assay was established in this thesis.

It was found in this thesis that deficiency in phototropin results in a delay of zygote germination under moderate light conditions. Intensive light input can overcome the lag caused by the low level of phototropin. In the interpretation of the result obtained from the mating assays, three factors are necessary to be placed under consideration. The first is how closely pab1 gene and aphVIII gene are linked in the Chlamydomonas genome i.e. are they on the same or different chromosomes. The second is the level of phototropin in transformant C4's offspring. The third is the presence of vegetative diploid cells.

Transformant C4 and control strain G5 were generated by transformation with aphVIII gene separately. aphVIII gene integrated into the Chlamydomonas genome randomly. Although there are 17 linkage groups in Chlamydomonas genome, there is still the possibility that in C4 or in G5, aphVIII fell into the same linkage group as the pab1 gene. The chance is high that aphVIII and pab1 are unlinked in both strains (16/17 × 16/17). The possibility is 2× 16/17 × 1/17 that in one strain the two genes were linked and in the other strain the two genes were in different linkage groups. It is also possible that in both strains, the two gene were linked (1/17 × 1/17). In this assay, aphVIII and pab1 are regarded as unlinked.

In those zygotes produced from the mating of C4 and CC124, there were two sets of chromosomes present in the nucleus. Whether the RNAi construct was transcriptionally active was unknown. In the vegetative cells produced from those zygotes, some contained the RNAi construct in their genome, some did not. How could the result be analyzed? The resolution was simple. As introduced in the introduction part, RNAi machinery takes place in cytoplasm. RdRp can use single-stranded siRNA as primer and target mRNA as template to synthesize double-stranded RNA. Those double-stranded RNAs can enter the cycle again. RNAi has a self-amplifying process. During zygote formation and meiosis, the siRNA left in the cytoplasm would trigger the process. Thus, the zygotes formed from C4 and CC124 were regarded as Phot1-.

The presence of vegetative diploid cells is another factor that should be considered in the data analysis. Under normal laboratory conditions, about 1-5% of mated gamete pairs of C. reinhardtii do not form meiotic zygotes. They form vegetative diploids (Harris, 1989; Ebersold, 1967) instead. The difference between germinated zygotes and vegetative diploid cells is that the diploid cells begin to divide shortly after mating whereas meiotic zygotes require several days to mature before germination (Harris, 1989). Thus the colonies of vegetative diploids came out earlier and were much bigger than those formed by germinated zygote. Those big colonies were not counted.

It is possible to remove the impact of vegetative diploids. On TAP medium, vegetative diploids can grow independently of the light conditions. Thus their number should be similar in high light condition, middle light condition and low light condition. They appeared as big colonies on plates. The number could just be removed from the colony numbers obtained under high light and middle light conditions.

By comparing the zygote germination situations between low light condition and middle light conditions or high light conditions, the conclusion that light is necessary for zygote germination could be drawn. By comparing the offspring of the transformant and the control strain in middle condition, it is concluded that phototropin is an important factor for zygote germination. Under certain light conditions, those zygotes with higher phototropin level would germinate faster. By comparing the offspring of the RNAi construct transformant in middle light and hight light conditions, the conclusion could be reached that strong light could compensate the lack of phototropin and accelerate zygote germination.

As an eukaryotic single-celled organism, C. reinhardtii has several advantages in expressing recombinant proteins over prokaryotic systems such as E. coli. 1.) With bioinformatic analysis, a total of 34 Chlamydomonas open reading frames, potentially encoding members of the five major chaperone families and the GrpE and Cpn10/20 co-chaperones, have been discovered (Schroda, 2004). Thus all the newly synthesized proteins have a milder folding surrounding compared with E. coli, which may greatly help the foreign peptides especially those from other eukaryotic systems to fold into their nature conformation. 2.) Chlamydomanas has its protein modification system, which could not only help to stabilize the protein conformation but also increase the protein's resistance against protease. 3.) The growth medium for this alga is quite simple and cheap. Cultures can be easily upscaled. This allows large cultures to be used as starting material when expression level of protein is low. But the drawbacks for Chlamydomonas as an expression system are also quite obvious. 1.) Foreign gene randomly integrates into the nuclear genome, when Chlamydomonas is transformed (Rochaix, 1995). This random integration could cause some unknown problem such as truncated expression or some other deleterious effect on the cell growth. 2.) Chlamydomonas cells can turn off the expression of foreign genes autonomously. The mechanism for such kind of silencing is still unknown (Cerutti et al., 1997). 3.) In most cases, cell wall deficient strains such as cw15 arg- A were chosen for transformation. Although they are easy to transform, it is difficult to grow them in large volume. They are not able to stand high pressure or shearing force and break in large volume culture, which could cause contamination easily.

The expression level of recombinant protein is decided by the transcriptional efficiency, degradation speed of mRNA, translation efficiency and speed of protein degradation. Thus, the strength of promoter is of great importance. The AR promoter (HSP70A promoter plus RbcS2 promoter) is a very strong promoter (Goldschmidt-Clermont and Rahire, 1986; Kindle, 1998; Schroda et al., 2000). In this thesis, AR promoter was not chosen to drive the expression of Ble gene fused with phototropin cDNA. It has been clearly demonstrated that insertion of the first intron of the RbcS2 gene into the Sh Ble coding sequence increased expression level around 30-fold when compared with RbcS2 promoter alone. The transcription driven by RbcS2 promoter need the aid from RbcS2 introns. Another strong promoter, PsaD promoter was chosen for driving the nucleic expression of the fusion product. The PsaD gene encodes an abundant protein of the Photosystem I complex and does not contain any introns in its coding sequence. The absence of introns in the PsaD gene suggests that the transcription driven by this promoter does not need additional information. The strength of the PsaD promoter is stronger than RbcS2 promoter alone and similar with RbcS2 plus first intron (Fisher and Rochaix, 2001).

Even with PsaD promoter, the trials to express phototropin cDNA met continuous failure (pLYM-D, pLYM-E in Chapter 3.4.3). Most of Chlamydomonas nuclear genes contain several small introns in the coding sequences and intronic sequences appear to have an important role in the regulation of gene expression (Silflow, 1998). Using genomic sequences in complementation of mutants seems to be more efficienct than their cDNA counterpart (Diener et al., 1993; Auchincloss et al., 1999; Perron et al., 1999; Boudreau et al., 2000). Studies of transgenes in higher eukaryotes, including plants, have demonstrated a positive role for introns in gene expression. In some cases, intron splicing appears to be required for efficient nuclear export or stability of transcripts (Huang and Gorman, 1990; Rose and Last, 1997). The introns of the phototropin gene could be important for transcription. It has not been tried to put several introns in the over-expression construct. Obviously, such attempt may result in some improvement (Eichler-Stahlberg, 2005).

Although the fusion expression strategy may not be the best way to express phototropin, it is the only method that currently works. Ble protects the cell by binding zeocin to prevent it from cutting DNA (Drocourt et al., 1990; Gatignol et al., 1988). To work efficiently, Ble proteins need to form dimers (Dumas et al., 1994). The presence of Strep tag II in its N-terminus without spacer seemed to hinder Ble part in the fusion product to form dimer (Figure 3.4.5.1.2).

From an widely accepted point of view, Ble protein needs to enter the nucleus to bind zeocin, thus the fusion expression products with Ble are required to be soluble. The fusion strategy with Ble has been tried in Chlamydmonas (Fuhrmann et al., 1999), where GFP was directly linked to the C-terminal of Ble. The fusion product was localized only in the nucleus (Fuhrmann et al., 1999). This finding suggests that there should be a nuclear localization signal (NLS) within the Ble protein sequence. The majority of phototropin attaches to the membrane, but a small portion of the protein remains soluble. The percentage of soluble protein in the expressed fusion product is similar to that of the original phototropin. This suggests that the mechanism which causes the insolubility of those two proteins could be same or closely related. The localization of the expressed fusion product is not determined in detail, and the mechanism of the fusion product helping the cells survive in presence of zeocin is unknown. The possible explanation could be the following: newly synthesized soluble fusion protein managed to enter the nucleus guided by the NLS of Ble. Those proteins remained soluble in nucleus and helped to protect the cell; during cell division, those proteins were released to the cytoplasm. When the new nucleus was formed, those soluble fusion proteins entered the nucleus again. Another explanation could be that the expressed protein formed a matrix attached to the plasma membrane and prevented zeocin entering the nucleus. However, further test such as fusing a GFP to the C-terminus of phototropin or isolating and fractionating nucleus should be carried out. Ble fused with kinase or LOV2 plus kinase domain could also be expressed in the same way, but almost 100% of the expressed product remained insoluble (data not shown), which support the matrix assumption.

The fusion expression strategy would be very useful for future studies of protein functional analysis in Chlamydomonas. And the method seems to have several advantages: 1.) Since the marker protein fused to the target protein, co-transformation is not required. 2.) The transformants are easy to screen. In Chapter 3.4.2, out of 80 clones, 66 showed the correct expression pattern, around 82% of the transformants were positive. 3.) The expression level of target protein could be regulated. Ble is a zeocin binding protein, the higher concentration of zeocin is used in the medium, the higher level of over-expression product is required for the cell. Thus, in a certain range, increased concentrations of the antibiotics can be used to identify those transformants which have high level of target protein. 4.) Transformants are not easily silenced. Normally introduced genes in Chlamydmonas are unstable in their expression level (Cerutti et al 1997). In this fusion strategy, those silenced cells could be easily killed by putting the transformants under selection pressure again. However, the safest way currently to keep Chlamydomonas transformants is still to freeze them in liquid nitrogen.

The fusion constructs encoding Ble -Kinase and Ble-LOV2-Kinase resulted in a low expression levels. Recently, Mastuoka and Tokutomi studied GST fused with kinase domain, GST fused with LOV2 - kinase construct and GST fused with LOV1-LOV2-kinase. When the kinase domain was expressed without LOV2 domain, kinase domain was always active. When LOV2 plus the kinase domain was expressed, the kinase domain got activated under low light conditions. When LOV1-LOV2-kinase was expressed, the kinase domain got activated only under high light conditions (Figure 4.4.1) (Mastuoka and Tokutomi, 2005 ). Although there is still debate whether LOV1 domain or LOV2 domain gets activated first, it was clearly demonstrated the presence of LOV1 and LOV2 domain helped to keep the kinase domain inactive in darkness or under low light conditions. This finding could explain the low expression level of Ble-kinase protein and Ble-LOV2-kinase protein in Chlamydomonas. Ble-kinase was permanently active and Ble-LOV2-kinase was easily activated. Thus, the over-expressed product might be harmful for the cells. They can only exist in low concentration.

It also could explain the failure of trials to over-express phototropin directly. Excess amount phototropin might be toxic to the cells under moderate light conditions. The expression level of recombinant phototropin is low. Thus, unconspicuous improvement in phototropin level would be hardly detected. The reason that the fusion expression product could be detected was due to the high recognition efficiency of anti Sh Ble antibody. The trials to express C.r. phototropin directly may be carried out in the future. Genomic PCR or RT-PCR should be applied in screening for the transformants. Strep tag II may be directly placed in the N-terminus or C-terminus of phototropin cDNA. The expressed product could be purified in the same way in Chapter 3.4.6.2.

Figure 4.4.1 Schematic illustration of the roles of LOV1 and LOV2 domains in light regulation of substrate phosphorylation by Kinase domain in A.t. Phot2. P: phosphate; S, substrate; LL, low light conditions; HH, high light conditions ( Mastuoka and Tokutomi, 2005).

It has been found that phototropin was very delicately regulated (Chapter 3.1). This regulation is consistent with the importance for growth and development of the cell. There are reports about success in expressing phototropin in phot1 null mutant (Sakamoto and Briggs, 2002) or phot1phot2 null mutant (Onodera et al., 2005) in Arabidopsis. However, success in over-expressing phototropin has not been reported. It is unknown whether excessive phototropin could be toxic for the cells. The reason for the success of fusion expression could be that Sh-Ble protein in the N-terminus of the fusion product blocked some proper functions of phototropin and made the cells less sensitive to the over expressed product.

An interesting transformant was found which only expressed part of the fusion product with better solubility. The missing of phototropin C-terminus may make the fusion product more hydrophilic and easy to purify. In the sequence analysis, around 50 aminoacids in the C-terminus of phototropin seem to be not necessary for the kinase activity (Huang et al., 2002), which suggest that this short fragment could be either modified or interacting with other proteins to form aggregates. Expression the 'truncated' version of the fusion product in Chlamydomonas might be a good approach to prepare material for protein crystallization.

As described in Chapter 3.1, in the cell wall deficient strain cw15 arg- A, under the same growth conditions, the amount of soluble phototropin is much less than that in the bald strain CC477. Different genetic background of the two strains could be the reason for that. cw15 arg- A is not suitable for growing of large scale cultures because of its fragile cell wall. In the future, bald strains are a good choice for expressing soluble phototropin for purification.