VL 6

• red light receptor in an aquatic organism? water absorbs red light

• in plants development (requierements: tissues!) absorption spectrum Phaedoactylum Phy1 after red/far illuminant

• higher absoprtion

• FR dependent genes

• signal transduction unknown

• specific for Heterkontae (first found in Xanthophyta, then studied in Diatoms)

• Blue light receptor - has LOV domain

• localised im nuleus

• light induced TF

• after light in a dimeric structure

• overepxoressed in E.coli

• supression of Lhcx expression

• CPF1 has dual function (photolyase and photoreceptor)

• same in Ostreococcus tauri (primitive green algae)

• CryP binds MTHF (antenna chromophore) and FADH* with long-wavelength red absorption

• in the dark oxidised to FAD

• photoreceptor function likely

• localised in nucleus

• CryP antisense mutants: about 50% of CryP protein remains

• WT and knock-down mutants (WT, NA1, TA3), 2-3 replicates

• 60nmin after BL after 72h dark adaptation or continuous dark

• 11-14 millions reads per sample

• 85% could be annotated

• Functional groups:

  -> 30% unknown

  -> in diatoms there are a lot of proteins of unknown function

• WT: many genes are regulated by blue light

• 310 genes differently expressed in both mutants vs WT

• regulated under BL > group 1

• regulated under BL and in darkness > group 2

• regulated only in darkness > group 3

• CPF1 and PHY expression are influenced by CryP

• CPF1: group 2 (BL as well as Dark)

• PHY: group 3

• CryP down-regulates mRNA level of Phy and CPF1 in the dark (late night)

• in addition the levels of CPF1 under BL (afternoon)

• high= oxixidises low=semired

-> FADH* extremely stable

• COP1 and SPA are missing in diatoms

• whole lane of eluate analysed per MS

• identified BoIA, ID42612, CSDP (and ribosomal subunits)

• via PHR (also in mutant w/o CTE)

• only to the dimer

• slight dependence on redox state of FAD

• tarsncription factor

• often stress induced

• fits to localisation of CryP

• fits to control of gene expression

• related to Bap31-proteins

• ER, mainly in nuclear ER

• in yeast involved in changes of gene expression, in plants miRNA binding

• fits localisation of CryP

• fits to gene expression control

• present in Heterokonts and relationship to Bap31 poor

• FCP

• lhcf = light harvesting function

• lhcr=light harvesting or protective function

• lhcx=photoprotection

• addition of His-tag for protein isolation

  • construction of plasmids

  • transformation of diatoms, waiting for ages…

  • characterisation of mutants

    > by molecular biology

    > physiologically

-> His-FCP binds selectively in trimers

• directly from the diatom in Cyclotella meneghiniana (Centrales)

  • batch cultures in inorganic medium

• thylakoid isolation:

  • cells broken via french press

  • several washing steps in low salt buffer

• solubilisation of the membrane proteins

• FCPa = trimers

• FCPb = nanonomers

• to PSII

• FCP are teramers in C. gracilis

• FCPa are also trimers T.pseudonana

• how are the thylakoids organized

• where are PSI, PSII and ATPase localised?

• what is the connection between the thylakoids?

• much stronger

• reduction of LHX1 (40-80%), no reduction in the pool of FCP

• no change in the PS/FCP ration

• only about 70% of the NPQ of WT

• Lhcx1 is needed for NP

• decrease in the fluoresecence amplitude in both FCPs and simmultaneously a shift tolonger wavelength

• both FCPs are principally able to quench

• FCPb less fluorescence (oligomer), independent of diatoxanthin

• FCPa more fluorescence (trimer), dependent on diatoxanthin

• less lipid per protein: smaller protein distance -> less fluorescence

• fluorescence yield dependent on pH

• pH-dependent quenching

• FCPa is acting like minor LHC and psbS in vascular plants:

• diatoxanthin depdent, pH-depndent and dependent on aggregation

• PCPb only dependet on aggregation

• lhcx1 is needed for NPQ

• in Cyclotella subunit of the trimers are probably pigmented

• precise molecular mechanism/switch for NPQ

• difference Pennales-centrales (regulation, FCP composition)

• similar absorption spectra of the FCP, no grana, no state transition

  -> how can the excitation energy distribution be regulated between photosystems?