Photoactivated adenylyl cyclase controls phototaxis in the flagellate Euglena gracilis

Abstract

Euglena gracilis, a unicellular freshwater protist exhibits different photomovement responses, such as phototaxis (oriented movement toward or away from the light source) and photophobic (abrupt turn in response to a rapid increase [step-up] or decrease [step-down] in the light fluence rate) responses. Photoactivated adenylyl cyclase (PAC) has been isolated from whole-cell preparations and identified by RNA interference (RNAi) to be the photoreceptor for step-up photophobic responses but not for step-down photophobic responses (M. Iseki, S. Matsunaga, A. Murakami, K. Ohno, K. Shiga, C. Yoshida, M. Sugai, T. Takahashi, T. Hori, M. Watanabe [2002] Nature 415: 1047-1051). The present study shows that knockdown of PAC by RNAi also effectively suppresses both positive and negative phototaxis, indicating for the first time that PAC or a PAC homolog is also the photoreceptor for photoorientation of wild-type E. gracilis. Recovery from RNAi occurred earlier for step-up photophobic responses than for positive and negative phototaxis. In addition, we investigated several phototaxis mutant strains of E. gracilis with different cytological features regarding the stigma and paraxonemal body (PAB; believed to be the location for the phototaxis photoreceptor) as well as Astasia longa, a close relative of E. gracilis. All of the E. gracilis mutant strains had PAC mRNAs, whereas in A. longa, a different but similar mRNA was found and designated AlPAC. Consistently, all of these strains showed no phototaxis but performed step-up photophobic responses, which were suppressed by RNAi of the PAC mRNA. The fact that some of these strains possess a cytologically altered or no PAB demonstrates that at least in these strains, the PAC photoreceptor responsible for the step-up photophobic responses is not located in the PAB.

Figure 1.

Anatomical features of the strains. E. gracilis wild type (A) with chloroplasts and normal PAB and stigma (S). Mutant strain FB (B) without chloroplasts and smaller than normal PAB and stigma. Astasia longa wild type and E. gracilis mutant strains 1F and 9F (C) also without chloroplasts and no detectable PAB and stigma.

Figure 2.

The 5′-end fragment of PACα (A) and PACβ (B) detected with the same set of primers by PCR in E. gracilis wild type (E.g.) and in mutant strains 1F, 9F, and FB. AlPACα and AlPACβ of A. longa (A.l.) were amplified with AlPAC specific primers.

Figure 3.

Tracks of cells at high irradiances summarized in circular histograms. Control cells swimming with high precision away from the light source (800 W m^-2 at 0°) and RNAi-treated cells showing random swimming.

Figure 4.

The effects of PAC on negative phototaxis of E. gracilis without RNAi (black squares) and after RNAi against PACα (white circles), PACβ (white triangles), and PACα&β (white squares). Silencing of PAC inhibited negative phototaxis as indicated by the decreased r value, an indicator of the precision of orientation.

Figure 5.

The effects of PAC on positive phototaxis of E. gracilis without RNAi (black squares) and after RNAi of each or both PAC subunit(s) (white symbols). Inhibition of positive phototaxis lasted for more than 2 months, similar to negative phototaxis.