A Musashi Splice Variant and Its Interaction Partners Influence Temperature Acclimation in Chlamydomonas.

Microalgae contribute significantly to carbon fixation on Earth. Global warming influences their physiology and growth rates. To understand algal short-term acclimation and adaptation to changes in ambient temperature, it is essential to identify and characterize the molecular components that sense small temperature changes as well as the downstream signaling networks and physiological responses. Here, we used the green biflagellate alga Chlamydomonas reinhardtii as a model system in which to study responses to temperature. We report that an RNA recognition motif (RRM)-containing RNA-binding protein, Musashi, occurs in 25 putative splice variants. These variants bear one, two, and three RRM domains or even lack RRM domains. The most abundant Musashi variant, 12, with a molecular mass of 60 kD, interacts with two clock-relevant members of RNA metabolism, the subunit C3 of the RNA-binding protein CHLAMY1 and the 5'-3' exoribonuclease XRN1. These proteins are able to integrate temperature information by up- or down-regulation of their protein levels in cells grown at low (18°C) or high (28°C) temperature. We further show that the 60-kD Musashi variants with three RRM domains can bind to (UG)7 repeat-containing RNAs and are up-regulated in cells grown at a higher temperature during early night. Intriguingly, the 60-kD Musashi variant 12, as well as C3 and XRN1, confer thermal acclimation to C. reinhardtii, as shown with mutant lines. Our data suggest that these three proteins of the RNA metabolism machinery are key members of the thermal signaling network in C. reinhardtii.

Antagonistic bacteria disrupt calcium homeostasis and immobilize algal cells.

Photosynthetic unicellular organisms, known as microalgae, are key contributors to carbon fixation on Earth. Their biotic interactions with other microbes shape aquatic microbial communities and influence the global photosynthetic capacity. So far, limited information is available on molecular factors that govern these interactions. We show that the bacterium Pseudomonas protegens strongly inhibits the growth and alters the morphology of the biflagellated green alga Chlamydomonas reinhardtii. This antagonistic effect is decreased in a bacterial mutant lacking orfamides, demonstrating that these secreted cyclic lipopeptides play an important role in the algal-bacterial interaction. Using an aequorin Ca2+-reporter assay, we show that orfamide A triggers an increase in cytosolic Ca2+ in C. reinhardtii and causes deflagellation of algal cells. These effects of orfamide A, which are specific to the algal class of Chlorophyceae and appear to target a Ca2+ channel in the plasma membrane, represent a novel biological activity for cyclic lipopeptides.

Novel interaction of two clock-relevant RNA-binding proteins C3 and XRN1 in Chlamydomonas reinhardtii.

The RNA-binding protein CHLAMY1 of the green alga Chlamydomonas reinhardtii consists of two subunits, named C1 and C3 that maintain the period and phase of the circadian clock. Here, we investigated if any of its subunits interact with other clock components involved in RNA metabolism. We found that C3, but not C1 strongly interacts with exoribonuclease XRN1 whose knockout results in low amplitude rhythms. XRN1 is subject to degradation by the proteasome pathway. Its level increases in cells grown at lower ambient temperature simulating night, which was also observed for C3. Our data indicate a network of clock-relevant RNA-binding proteins.

How the green alga Chlamydomonas reinhardtii keeps time.

The unicellular green alga Chlamydomonas reinhardtii has two flagella and a primitive visual system, the eyespot apparatus, which allows the cell to phototax. About 40 years ago, it was shown that the circadian clock controls its phototactic movement. Since then, several circadian rhythms such as chemotaxis, cell division, UV sensitivity, adherence to glass, or starch metabolism have been characterized. The availability of its entire genome sequence along with homology studies and the analysis of several sub-proteomes render C. reinhardtii as an excellent eukaryotic model organism to study its circadian clock at different levels of organization. Previous studies point to several potential photoreceptors that may be involved in forwarding light information to entrain its clock. However, experimental data are still missing toward this end. In the past years, several components have been functionally characterized that are likely to be part of the oscillatory machinery of C. reinhardtii since alterations in their expression levels or insertional mutagenesis of the genes resulted in defects in phase, period, or amplitude of at least two independent measured rhythms. These include several RHYTHM OF CHLOROPLAST (ROC) proteins, a CONSTANS protein (CrCO) that is involved in parallel in photoperiodic control, as well as the two subunits of the circadian RNA-binding protein CHLAMY1. The latter is also tightly connected to circadian output processes. Several candidates including a significant number of ROCs, CrCO, and CASEIN KINASE1 whose alterations of expression affect the circadian clock have in parallel severe effects on the release of daughter cells, flagellar formation, and/or movement, indicating that these processes are interconnected in C. reinhardtii. The challenging task for the future will be to get insights into the clock network and to find out how the clock-related factors are functionally connected. In this respect, system biology approaches will certainly contribute in the future to improve our understanding of the C. reinhardtii clock machinery.