D in menaquinone biosynthesis in bacteria.b2016 The Authors. The Plant
D in menaquinone biosynthesis in bacteria.b2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley Sons Ltd., The Plant Journal, (2017), 89, 141Loss of phylloquinone in Chlamydomonas 143 seedling-lethal phenotype (Kim, 2008). In contrast, the Arabidopsis menG-homologous deficient mutant is viable since, as in Synechocystis, demethylphylloquinone acts as substitute for PhQ in PSI (Lohmann et al., 2006). In 2015 it was established in Synechocystis and Arabidopsis that the biosynthesis of PhQ has an extra step: the reduction from the demethylphylloquinone ring by a type-II NADPH dehydrogenase, known as NdbB in Synechocystis and NDC1 in Arabidopsis, before its trans-methylation by MenG (Fatihi et al., 2015). Synechocystis ndbB and Arabidopsis ndc1 mutants display elevated photosensitivity to high light like the PhQ-deficient mutants previously characterized in these organisms. Inside the green alga C. reinhardtii, which can be a model organism for studying the photosynthetic machinery (Hippler et al., 1998), characterization in the PhQ biosynthetic pathway is still incomplete. Up to now, only 1 mutant, deficient for MEND IL-8/CXCL8 Protein site protein, has been characterized (LefebvreLegendre et al., 2007). Inactivation of MEND in C. reinhardtii, as in Synechocystis sp. PCC 6803 (Johnson et al., 2003), results in the full loss of PhQ and its replacement by PQ in PSI. Nonetheless, accumulation of PSI is not affected in this mutant and also the absence of PhQ rather causes a decrease inside the size of your PQ pool and of synthesis of PSII subunits. The phenotype of the only mutant isolated in C. reinhardtii is hence neither close for the one particular described in cyanobacteria or to that of land plants. This observation prompted us to isolate new mutants of the PhQ biosynthetic pathway in C. reinhardtii. In anoxia, a double reduction of PQ into PQH2 in the A1 website occurs LAIR1 Protein Source Within the mend mutant, interrupting photosynthetic electron transfer (Lefebvre-Legendre et al., 2007; McConnell et al., 2011). Within this operate, we took advantage of this photosynthetic deficiency in anoxia to isolate four new Chlamydomonas mutants affected in either the MENA, MENB, MENC or MENE enzymatic step on the PhQ biosynthesis pathway. Results A peculiar chlorophyll induction curve is specific for identification of PhQ-deficient mutants Nine sequences corresponding to nine in the ten enzymatic actions required for the PhQ biosynthesis pathway in cyanobacteria and land plants may be located inside the C. reinhardtii genomic database (v.5.5 on PHYTOZOME) (Table 1). Genomic sequences coding for MENF, MEND, MENC and MENH enzymatic domains are positioned in a single open reading frame (ORF), and are named PHYLLO by similarity to gene organization inside a. thaliana (Gross et al., 2006), and likely coding for any tetramodular enzyme. We did not uncover any homolog to the DHNA-CoA thioesterase performing the seventh step from the pathway in cyanobacteria and land plants but a putative candidate (TEH4) is suggested (see Discussion). To isolate new C. reinhardtii strains deficient in PhQ biosynthesis we screened 13 250 hygromycin-resistant (HygR) transformants and 3500 paromomycin-resistant (ParR) transformants by an in vivo chlorophyll fluorescence imaging screening protocol. The screening process is based on the observation that a double reduction of PQ in PQH2 within the A1 website happens inside a mend mutant in anoxia, interrupting photosynthetic electron transfer (McConnell et al., 2011). We hence recorded the ch.