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The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.

Cyanobacteria are promising photosynthetic organisms owing to their ease of genetic manipulation. Among them, Synechococcus elongatus UTEX 2973 exhibits faster growth, higher biomass production efficiency and more robust stress tolerance compared with S. elongatus PCC 7942. This is due to specific genetic differences, including four single-nucleotide polymorphisms (SNPs) in three genes. One of these SNPs alters an amino acid at position 252 of the FoF1 ATP synthase α-subunit from Tyr to Cys (αY252C) in S. elongatus 7942. This change has been shown to significantly affect growth rate and stress tolerance, specifically in S. elongatus. Furthermore, experimental substitutions with several other amino acids have been shown to alter the ATP synthesis rate in the cell. In the present study, we introduced identical amino acid substitutions into Synechocystis sp. PCC 6803 at position 252 to elucidate the amino acid's significance and generality across cyanobacteria. We investigated the resulting impact on growth, intracellular enzyme complex levels, intracellular ATP levels and enzyme activity. The results showed that the αY252C substitution decreased growth rate and high-light tolerance. This indicates that a specific bulkiness of this amino acid's side chain is important for maintaining cell growth. Additionally, a remarkable decrease in the membrane-bound enzyme complex level was observed. However, the αY252C substitution did not affect enzyme activity or intracellular ATP levels. Although the mechanism of growth suppression remains unknown, the amino acid at position 252 is expected to play an important role in enzyme complex formation. [ABSTRACT FROM AUTHOR]

The mechanisms governing chemotaxis in Chlamydomonas reinhardtii are largely unknown compared to those regulating phototaxis despite equal importance on the migratory response in the ciliated microalga. To study chemotaxis, we made a simple modification to a conventional Petri dish assay. Using the assay, a novel mechanism governing Chlamydomonas ammonium chemotaxis was revealed. First, we found that light exposure enhances the chemotactic response of wild-type Chlamydomonas strains, yet phototaxis-incompetent mutant strains, eye3-2 and ptx1, exhibit normal chemotaxis. This suggests that Chlamydomonas transduces the light signal pathway in chemotaxis differently from that in phototaxis. Second, we found that Chlamydomonas collectively migrate during chemotaxis but not phototaxis. Collective migration during chemotaxis is not clearly observed when the assay is conducted in the dark. Third, the Chlamydomonas strain CC-124 carrying agg1−, the AGGREGATE1 gene (AGG1) null mutation, exhibited a more robust collective migratory response than strains carrying the wild-type AGG1 gene. The expression of a recombinant AGG1 protein in the CC-124 strain suppressed this collective migration during chemotaxis. Altogether, these findings suggest a unique mechanism; ammonium chemotaxis in Chlamydomonas is mainly driven by collective cell migration. Furthermore, it is proposed that collective migration is enhanced by light and suppressed by the AGG1 protein. [ABSTRACT FROM AUTHOR]

Phosphoribulokinase (PRK), one of the enzymes in the Calvin–Benson cycle, is a well-known target of thioredoxin (Trx), which regulates various enzyme activities by the reduction of disulfide bonds in a light-dependent manner. PRK has two Cys pairs conserved in the N-terminal and C-terminal regions, and the N-terminal one near the active site is thought to be responsible for the regulation. The flexible clamp loop located between the N-terminal two Cys residues has been deemed significant to Trx-mediated regulation. However, cyanobacterial PRK is also subject to Trx-dependent activation despite the lack of this clamp loop. We, therefore, compared Trx-mediated regulation of PRK from the cyanobacterium Anabaena sp. PCC 7120 (A.7120_PRK) and that from the land plant Arabidopsis thaliana (AtPRK). Interestingly, peptide mapping and site-directed mutagenesis analysis showed that Trx was more effective in changing the redox states of the C-terminal Cys pair in both A.7120_PRK and AtPRK. In addition, the effect of redox state change of the C-terminal Cys pair on PRK activity was different between A.7120_PRK and AtPRK. Trx-mediated redox regulation of the C-terminal Cys pair was also important for complex dissociation/formation with CP12 and glyceraldehyde 3-phosphate dehydrogenase. Furthermore, in vivo analysis of the redox states of PRK showed that only one disulfide bond is reduced in response to light. Based on the enzyme activity assay and the complex formation analysis, we concluded that Trx-mediated regulation of the C-terminal Cys pair of PRK is important for activity regulation in cyanobacteria and complex dissociation/formation in both organisms. [ABSTRACT FROM AUTHOR]

Redox-based regulatory systems are essential for many cellular activities. Chlamydomonas reinhardtii exhibits alterations in motile behavior in response to different light conditions (photokinesis). We hypothesized that photokinesis is signaled by variations in cytoplasmic redox poise resulting from changes in chloroplast activity. We found that this effect requires photosystem I, which generates reduced NADPH. We also observed that photokinetic changes in beat frequency and duration of the photophobic response could be obtained by altering oxidative/reductive stress. Analysis of reactivated cell models revealed that this redox poise effect is mediated through the outer dynein arms (ODAs). Although the global redox state of the thioredoxin-related ODA light chains LC3 and LC5 and the redox-sensitive [Ca.sup.2+]-binding subunit of the docking complex DC3 did not change upon light/dark transitions, we did observe significant alterations in their interactions with other flagellar components via mixed disulfides. These data indicate that redox poise directly affects ODAs and suggest that it may act in the control of flagellar motility.