2.3 Light-regulated behavior in white collar mutants
2.3.1 white collar mutants synchronize conidiation in response to light
E 1 2 3 4 5 6 7 12 24 0 A B 1 2 3 4 5 6 7 C 1 2 3 4 5 6 7 wt wc-1RIP#7 wc-2234w wc-1RIP#7 wc-2234w D 1 2 3 4 5 6 7 frq10 wt wc-1RIP#7 wc-2234w wc-1RIP#7 wc-2234w
Figure 22. Conidiation in w c - 1 and wc-2 deficient strains is synchronized in LD cycles. Data from a week of 12:12 LD cycles (12 h light 12 h dark) are double plotted (two days per line, repeating the day graphed on the right side on the left side of the next line down). The dark period is indicated by gray shading. The peaks on the graphs represent consolidation of conidiation, and the corresponding Neurospora race tubes are shown in (E). A. wild type (wt); B. wc–1RIP#7
(Talora et al., 1999) C.
wc–2234W (Linden & Macino 1997); D. wc–1RIP#7wc–2234W double mutant;
E. Race tubes for the above graphs and also for frq10
(only a race tube is shown for this strain, as it fails to synchronize conidiation to light’; Merrow et al., 1999). Fluence rates for A–C are 4 µE m–2
sec–1
, for D and E, 35 µE m–2
sec–1
. The race tube shown for wt runs for longer than the 7 days shown on the graph; the loss of synchrony seen in the last day of graph (C) reflects early removal of the race tubes from experimental conditions (as published in Dragovic et al. 2002).
In the experiments described here, a robust, light–regulated physiology is seen for both wc mutants, with conidia production consolidated to a discrete phase of the LD cycle (Fig. 22B–C), qualitatively similar to the wild type strain (Fig. 22A). In the wt the phase angle of conidiation (measured relative to onset of the conidial band) occurs in the middle of the night. The phase of conidiation in the mutant strains is, however, delayed relative to wild type. In wc–1RIP#7
, the onset of conidiation is delayed for ~2.5 h. In wc–2234W
, the phase of conidiation is delayed even further, for 3 h.
Wc–1RIP#7
is blind for most of the same outputs as wc–2234W
. Several scenarios accommodate these observations, including the possibility that one or both protein products act as photoreceptors. Given the observation that either of the single mutants can synchronize conidiation to light, a double mutant, wc–1RIP#7wc–2234W, was generated
and submitted to 24 h LD cycles. Like the single mutants, the double mutant strain consolidates conidia production in the light phase (Fig. 22 D and E), with a phase similar to the single mutants (delayed by 2.8 h relative to wild type).
Figure 23. Appearance of conidial bands in different media. Race tubes from a 12:12 LD cycle are shown. Both wt and wc-1RIP#7
mutant consolidate conidiation in response to L:D cycles on different media. All three media contain 1X Vogel’s salts, 0.5% Arginin but the carbon source is different : 0.3% Glucose (A), 0.3% Qunic acid (B) or no carbon source (C).
Nutrition has been shown to play a role in light reception (Nakashima and Fujimura, 1982; Sokolovsky et al., 1992) and clock mutants do not have the nutritional compensation that wild type strains have (Loros and Feldman, 1986). Therefore, several media formulations were compared for appearance of conidial bands in the mutant strains. Media containing either glucose, quinic acid (a carbon source encountered in nature by Neurospora) or no carbon source was tested. Although responses to light are clearly seen in cycles run under all three conditions, the bands were clearest on media with quinic acid or with no carbon source (Fig. 23). The bands produced by the mutant strains increase in density over the course of the week–long race tube experiment, similar to what is seen for the free–running rhythm in FRQ–less strains (Loros et al., 1986). Thus, long race tubes were used to improve data acquisition possibilities.
wc-1RIP#7 12 hours 24 0 A averageconidiation 1 2 3 4 5 6 7 12 hours 24 0 averageconidiation 1 2 3 4 5 6 7 averageconidiation 1 2 3 4 5 6 7 B 12 hours 24 0 averageconidiation 1 2 3 4 5 6 7 C wc-1RIP#21 wc-1KO#131
Figure 24. Conidiation in various wc-1 mutantsis synchronized in LD cycles. Data from a week of 24 h LD cycles, are double plotted. The dark period is indicated by gray shading. The peaks on the graphs represent consolidation of conidiation. The fluence rate for all race tubes was 35 µE m–2
sec–1
. (A) wc–1RIP#7
strain with RIPed promotor (see Methods) and 5’ end of ORF (Talora et al., 1999) ; (B) wc–1RIP#21
, RIP of entire ORF ( He at al. 2002); (C) In the wc–1KO#131
the wc-1 ORF is replaced by the hph gene (Lee at al., 2002).
Figure 25. Conidiation in various wc-2 deficient strains is synchronized in LD cycles. The fluence rate for all race tubes was 20 µE m–2
sec–1
(75% cool fluorescent white light Osram L36 and 25% was UVA light Osram L80 solarium light bulb). The wc–2234w
strain is point mutant that results in the production of a truncated protein (234w; FGSC number 3187); In the wc–2KO#99
the wc-2 ORF is replaced by a hph gene (Collett et al., 2002). (A) Data from a week of 24 h LD cycles are double plotted. (B) Race tubes corresponding to double-plotted data.
To distinguish if light-regulated conidiation is a general property of wc-1 deficient strains, additional wc mutants were assayed for entrainment. We have employed two wc–1 strains from independent sources. wc–1RIP#21
is mutant with a non-functional wc-1 allele, generated by the introduction of stop codons throught the entire ORF by RIP (Y. Liu personal communication and He et al., 2002; Fig. 24B). wc–1KO#131
was made by replacing the wc-1 ORF-segment encoding amino acids 59-1133 with the gene for hygromycin resistance (hph, hygromycin B phosphotransferase; Lee et al., 2003). The
wc–1RIP#21 and wc–1KO#131 strains (Fig. 24B and 24C) also consolidate conidiation in the
light phase with the phase delayed by 2-3 h relative to wc-1RIP#7
. The light source was cool fluorescent light (75%) enriched for UVA wavelengths with solarium light (25%, see also legend for Fig. 24 and Methods).
Additional wc-2 mutants were also assayed for entrainment in light cycles. wc–2234w
mutant (FGSC#3817) is a point mutant with premature stop codon, creating a truncated protein of 356 amino acids. Given the presence of a truncated WC–2 protein, and despite the lack of light–induced carotenoids, the light response in the wc-2234w
mutant could be due to residual activity deriving from the N terminus of the protein. To confirm that the light–induced behavior of the wc-2234w
mutant does not derive from this protein fragment, a complete wc–2 knockout wc-2KO#99
(Collett et al., 2002) was tested in the same experiments and found to regulate conidiation according to the LD cycle (Fig 25). Race tubes (Fig 25A) were analyzed and data are double plotted in Fig. 25B. In the