Expression of genes and division of labour between NEP and PEP in Plastids
The first model that was established concerning the functioning of NEP and PEP in plastids was the so-called “Cascade Model” which stated that at the early stage of chloroplast development, NEP transcribes house keeping genes i.e. genes encoding ribosomal proteins and genes encoding subunits of PEP in order to synthesize ribosomes and the PEP polymerase. According to this model, NEP is preferentially active during early stages of plastid development. With the advancement of chloroplast development, PEP takes over transcription and transcribes preferentially the photosynthesis related genes. It was further proposed that, switching from NEP to PEP activity during chloroplast development implicates inactivation of RpoTp by transfer RNA glutamate (tRNAglu) (Hanaoka et al., 2005) and sequestration of RpoTmp to thylakoid membranes (Azevedo et al., 2008). During the establishment of photosynthetically competent chloroplasts, PEP transcribes photosynthesis related genes that are then differentially regulated by light (Mullet, 1993).
The “Cascade Model” was later challenged by the findings that 1) NEP and PEP are present in mature chloroplasts (Bligny et al., 2000), 2) the activities of both the NEP and PEP increase with chloroplast development in maize and 3) PEP is active even during germination i.e. in differentiating protoplasts and is important for efficient germination of seeds of Arabidopsis (Demarsy et al., 2006). From these results our group has proposed a new model of “Co-existence of PEP and NEP” during germination.
The transcriptional apparatus of Chlamydomonas reinhardti chloroplast
The chloroplast of Chlamydomonas has a different transcriptional machinery and arrangement of genes in transcriptional units when compared to higher plant plastids. Complete absence of transcription in PEP inhibited plastids (Guertin & Bellemare, 1979), the failure to obtaine PEP knock out mutants (Fischer et al., 1996; Goldschmidt-Clermont, 1991) and the lack of tagetitoxin resistant transcription in plastids (Lilly et al., 2002) indicated that PEP is indispensable for plastid transcription and that there is no phage type nucleus encoded RNA polymerase. Only one nuclear RpoT gene was found which codes for a mitochondrion localized RNA polymerase. Most of the plastidial promoters do not have the -35 box but posses an elongated – 10 box. Surzycki & Shellenbarger (1976) at first reported a σ-like activity in chloroplasts. Immunological evidence of σ -like factors was then obtained by Troxler et al. (1994). Later on it was found that chlamydomonas posses only one gene (CrRpoD) encoding a σ -like factor (Bohne et al., 2006; Carter et al., 2004). Lilly and collaborators carried out a detailed northern blot, microarray and inhibitor analysis of the genome wide transcription in chlamydomonas chloroplasts and found that 1) sulfate deficiency results in 2-10 fold reversible decrease in transcription. 2) phosphate limitation results in 2-3 fold increase in transcript accumulation and 3) chloroplast lacks a nucleus encoded RNA polymerase (Lilly et al., 2002).
Role of sigma factors in plastid transcription of higher plants
AtSIG1 was at first characterized by Privat et al. (2003) in Arabidopsis by using an anti sense approach. The mutant plants did not show any visible phenotype. A T-DNA insertion mutant for sigma1 were later on characterised in Oryza sativa (Tozawa et al., 2007). OsSIG1 plays an important role in the chloroplast transcription of the genes of three different operons, psaA, psbB, and psbE. The reduction of transcripts was 68-89% for the psaA operon that consists of psaA, psaB and rps14; 41-48 % for the psbB operon (psbB, psbT, psbH, petB and petD) and 15-25 % for the psbE (psbE, psbT, psbL and psbJ) operon in sig1 plants. Thus sigma1 regulates PSI and PSII gene expression in rice. The genes whose transcript accumulation was increased in Ossig1 plants were rpl22, rpoA, rpoB, rpoC1, petE, petA, psbG, ORF159, psbZ and psbI. Northern blot analysis showed that the 5.2 Kb long transcript of psaA which is a tri-cistronic transcript was 46-49 % reduced in mutant plants. Similarly, the 2 Kb transcript of psbB was 23-34 % reduced in mutant plants while the 0.9 Kb transcript of psbE was reduced for 10-18 %. In contrast, the 2.6 Kb transcript of atpB (di-cistronic) was increased by 25-40%. The 1.9 Kb transcript of rbcL was present in the same quantities in wild type and mutant plants.
The western blot analysis showed that the PSI reaction center complex (PSAA-PSAB) was reduced by 26% in Ossig1 plants and also the activity of PSI was reduced. However, mutant plants have a functional PSII and the reduction in the electron transfer through PSII is probably due to the defect in PSI. Mature leaves of sig1 mutants have 1/3rd reduction in chlorophyll contents.
Using the yeast two hybrid system, a protein binding to the region 4.0 of SIGMA1 was found. This protein was named SibI (Morikawa et al., 2002). As it is known that region 4.2 is important for recognition of the -35 elements (Campbell et al., 2002), it was speculated that SibI may play an important role in modifying SIG1 promoter preference or regulating its activity. But experimental proof for this hypothesis is still lacking.
Recently, it was found that redox signals regulate the phosphorylation of SIG1 which in turn inhibits specifically the transcription of psaA gene. It was found that Thr-170 of SIG1 is phosphorylated. Under oxidative conditions of plastoquinone (PQ) the amount of phosphorylated SIG1 is increased (Shimizu et al., 2010, Lerbs-Mache, 2011). The authors found that phosphorylation of SIG1, through psaA gene expression, plays an important role in regulating the stoechiometry of PS-I and PS-II.
The expression of SIGMA2 is induced by red light as well as blue light (Mochizuki et al., 2004). A T-DNA insertion mutant for SIG2 shows pale green cotyledons and poor growth (Kanamaru et al., 2001). There was 15 % reduction in chlorophyll content in the mutant plants. The chloroplast number was the same as in wild type plants but plastids were smaller in size and the internal structures were poorly developed. Sig2 plants showed reduced level of transcript accumulation of four out of the six tRNAs that had been analysed i.e. trnV-UAC, trnM-CAU, trnE-UUC, trnD-GUC. The transcript level of trnG-GCC and trnW-CCA remained unchanged. On the protein level, accumulation of the D1 subunit of PSII, cytochrome f, the β subunit of chloroplast coupling factor 1, Rubisco Large subunit, a catalytic subunit of clpP protease and an acetyle co-enzyme A Carboxylase subunit was reduced in sig2-1 plants as compared to that in wild type plants (Kanamaru et al., 2001). The above described decrease in tRNAs might explain the strong reduction in protein accumulation. In addition to tRNAs, one of the three primary transcripts (starting at position – 256 from the ATG translation initiation codon) of psbD was also highly reduced in sig2 plants. However, the -946 primary transcript of psbD was more accumulated in sig2-1 plants as compared to that in wild type plants (Kanamaru et al., 2001). A SIG2 transcriptome analysis was performed by microarray and showed that on the mRNA level only the psaJ transcript was reduced in sig2-1 plants. Transcripts of the other 47 genes were increased (Nagashima et al., 2004a). Most of the genes whose expression/transcript accumulation was increased in sig2 plants were predominantly transcribed by NEP.
Sequence alignment of plant sigma factors with the E.coli primary sigma factor σ70 showed that SIG2 and SIG6 have the highest homology (Privat et al., 2003). AtSIG2 was also characterized in Arabidopsis by using an anti sense approach (Privat et al., 2003). SIG2 anti sense plants were deficient in chlorophyll during early stages of development (white cotyledons) and there was an efficient recovery of the wild type phenotype in later developmental stages. At later developmental stages, the phenotype of sig2 anti sense plants was different from that observed in T-DNA knock out plants (Kanamaru et al., 2001). In SIG2 anti sense plants, chlorophyll deficiency was restricted to cotyledons while in T-DNA knock out plants the chlorophyll deficiency is found in leaves as well i.e. it continues till later stages of development. Two hypotheses were raised by the authors to explain the recovery of wild type phenotype in mutant plants. 1) Expression of SIG2 is regulated at the post transcriptional level and SIG2 deficiency is compensated by protein stability in later stages of development (Privat et al., 2003). 2) Phenotype recovery at later stages of development may be explained also because one of the other sigma factors may take over the role of SIG2 for promoter specificity and transcription. SIG3 was proposed as a strong candidate for substitution of SIG2 as it has a similar promoter specificity (Hakimi et al., 2000; Privat et al., 2003) and SIG3 protein is increased in the absence of SIG2 (Privat et al., 2003).
SIG2 is a soluble protein. Interestingly SIG2 protein is more abundant in cotyledons than in leaves while SIG3 protein is more abundant in leaves than in cotyledons. This observation could explain why SIG3 can substitute SIG2 more easily in leaves than in cotyledons (Privat et al., 2003). So, SIG2 might play a key role in cotyledons while SIG3 is required for transcription in leaves.
An anti sense mutant of AtSIG3 was characterized in arabidopsis by Privat et al., (2003) and no visible phenotype was detected. Later on, two T-DNA insertion mutants in SIG3 (sig3-2, sig3-4) from the SALK collection were characterised (Zghidi et al., 2007). Sig3-2 has an insertion at the border of intron 1 and exon 2 while sig3-4 has an insertion within exon 4. Both of these mutants showed no visible phenotype, thus confirming the result of anti sense plants. Microarray analysis showed a strong reduction of the transcript level of psbN and a moderate reduction of the transcript level of all atp genes except for atpI. The psbN gene is located on the opposite strand of the psbB operon in the intergenic region between psbT and psbH. Primer extension and TAP RACE analysis showed that the promoter of psbN is located at position -32 from the ATG translation initiation codon of psbN and transcription initiation is totally under control of PEP SIG3 holoenzyme.
Table of contents :
1. EXPRESSION ANALYSIS OF THE TWO PLASTID ATP OPERONS: THE LARGE ATPI/H/F/A AND THE SMALL ATPB/E OPERON.
1.1. Transcriptional analysis of atpI transcripts.
1.2. Transcriptional analysis of atpH transcripts.
1.2.1. 3’ end mapping of -418 initiated atpH transcripts.
1.2.2. 3’ end mapping of -45 processed atpH transcripts.
1.2.3. Regulation of the higher stoichiometry of ATPH.
1.3. Transcriptional analysis of atpF transcripts
1.4. Transcriptional analysis of atpA transcripts.
1.5. Transcriptional analysis of atpB transcripts.
1.6. Expression analysis of the atpE gene.
2. DO LIGHT CONDITIONS INFLUENCE THE EXPRESSION OF SIG3 DEPENDENT GENES?
2.1. Illumination of etiolated plantlets.
2.1.1. Macroarray analyses
2.1.2. Action of light on SIG3 dependent gene expression Conclusion on the macroarray results:
2.1.3. Primer extension analyses
2.2. Light stress of green plants (photoinhibition of chloroplasts). Conclusion:
3. EXPRESSION ANALYSIS OF THE PSBT SENSE/ANTISENSE RNAS.
3.1 Previous results obtained in the laboratory.
3.2 Mapping of psbT anti sense RNA extremities.
3.3 Mapping of psbT sense RNA extremities.
3.4 Putative role of psbN expression on processing of psbB operon.
3.5 Existence of an internal psbT promoter within psbB gene.
Expression analysis of the two plastid encoded ATPsynthase operons: the large ATPI/H/F/A and the small ATPB/E operon.
Influence of light on the expression of SIG3 dependent transcripts
Expression analysis of the psbT sense/antisense RNAs.
MATERIAL AND METHODS :
Cultivation of plants in vitro:
Cultivation of plants in soil:
Extraction of RNA:
Treatment of RNAs with DNase:
Northern Blot analysis:
Preparation of polyacrylamide gel:
Cloning of the DNA Fragment:
Table of contents
Miniprep; Plasmid DNA Extraction:
RNA Analysis by RT-PCR:
Analysis of plastidial transcript profile expression by cDNA macroarray:
Hybridisation of labelled cDNAs on membrane:
Analysis of Results:
TAP (Tobacco Acid Pyrophosphate) treatment and 5’ RACE:
Circular RT- PCR:
Western Blot Analysis:
Western Blot analysis for smaller proteins