Edward D. Lipson

MODIFIED ACTION SPECTRA OF PHOTOGEOTROPIC EQUILIBRIUM IN Phycomyces blakesleeanus MUTANTS WITH DEFECTS IN GENES madA, madB, madC, and madH

Abstract— Action spectra of photogeotropic equilibrium were measured for behavioral mutants of Phycomyces blakesleeanus with defects in the genes madB, madC and madH as well as for a double mutant defective in the genes madA and madC. The action spectra of strains C109 (madB), LI (madC) and L52 (madA madC) all lack the broad near‐ultraviolet peak which extends from 347 to 386 nm in the wild type; the peaks at 414 and 491 nm are also missing in these mutants. The double mutant L52 (madA madC) shows a novel broad peak at 477 nm; the relative quantum effectiveness of L52 at 477 nm is 10 times higher than in LI (madC119). These properties of the double mutant L52 (madA madC) suggest steric interaction of the madA and the madC gene products in the photoreceptor complex. For the hypertropic mutant L84 (madH) the action spectrum and absolute sensitivity are similar to those for wild type. These results confirm and extend previous findings that multiple photoreceptors are mediating phototropism in P. blakesleeanus.

Photoinduced accumulation of carotene in Phycomyces

Blue light stimulates the accumulation of beta-carotene (photocarotenogenesis) in the fungus Phycomyces blakesleeanus. To be effective, light must be given during a defined period of development, which immediately precedes the cessation of mycelial growth and the depletion of the glucose supply. The competence periods for photocarotenogenesis and photomorphogenesis in Phycomyces are the same when they are tested in the same mycelium. Photocarotenogenesis exhibits a two-step dependence on exposure, as if it resulted from the additon of two separate components with different thresholds and amplitudes. The low-exposure component produces a small beta-carotene accumulation, in comparison with that of dark-grown mycelia. The high-exposure component has a threshold of about 100 J· m−2 blue light and produces a large beta-carotene accumulation, which is not saturated at 2·106 J·m−2. Exposure-response curves were obtained at 12 wavelengths from 347 to 567 nm. The action spectra of the two components share general similarities with one another and with those of other Phycomyces photoresponses. The small, but significant differences in the action spectra of the two components imply that the respective photosystems are not identical. Light stimulates the carotene pathway in the carB mutants, which contain the colourless precursor phytoene, but not beta-carotene. Carotenogenesis is not photoinducible in carA mutants, independently of their carotene content. This and other observations on various car mutants indicate that light prevents the normal inhibition of the pathway by the carA and carS gene products. The chromophore(s) for photocarotenogenesis are presumably flavins, and not carotenes.

ACTION SPECTRA FOR PHOTOTROPIC BALANCE IN Phycomyces blakesleeanus: DEPENDENCE ON REFERENCE WAVELENGTH and INTENSITY RANGE

Abstract— Action spectra for phototropic balance of Phycomyces blakesleeanus sporangiophores were measured for various reference wavelengths and intensity ranges. Balance action spectra were made at fluence rates of 10‐4 W m‐2 with reference wavelengths of 450 nm, 394 nm, 507 nm, and broadband blue light. For broad‐blue light and 450 nm light as references, typical flavin‐like action spectra were found with a ma jor peak at 455 nm, a secondary peak at 477 nm, and a minor peak at 383 nm; these peaks are wider for broad blue than for 450 nm light. With the 394 nm reference, there is a major peak at 455 nm, a secondary peak at 477 nm and a minor peak at 394 nm. An action spectrum with 507 nm reference has a major peak at 455 nm and a minor peak at 383 nm, but no peak at 477 nm. A balance action spectrum was made with 450 nm reference light near threshold intensity (2 times 10‐8 W m‐2); there, the 386 nm peak is greatly reduced, while the 455 nm peak is enhanced. The intensity dependence of the 386 nm peak was studied in detail for reference light of 450 nm. We found that the relative quantum efficiency of the 386 nm light increases with the logarithm of the 450 nm fluence rate; in the high intensity range (0.3 W m‐2) the relative quantum efficiency of the 386 nm light is 1.3 and approaches zero at 10‐9 W m‐2. These findings indicate that P. blakesleeanus phototropism is mediated by multiple interacting pigments or by a photochromic photoreceptor.

Double mutants of Phycomyces with abnormal phototropism

SummarySeven genes (madA to madG) are known which effect phototropism in Phycomyces. These genes have been partially ordered with respect to the associated stimulus-response pathway. Mutants affected in these genes serve as useful probes of photosensory transduction processes in this model system. To extend and deepen the analysis of the system, we have constructed a family of 21 double mutants in all combinations for the seven mad genes. A set of seven standard alleles was adopted for this work. The double mutants were isolated from crosses between isogenic single-mutant strains of opposite mating type. After a partial physiologic screening of the progeny, the double mutants were identified by complementation tests using single-mutant strains of known genotype. For all but three of the double mutants, the photogeotropism phenotypes were distinct from those of the respective single-mutant parentals. One triple mutant (madA madB madC) was constructed as part of this work. Various applications of the double mutants and the triple mutant are discussed. Recombination analyses were performed on the progeny from seven mad crosses to complete an earlier study. The results establish that all seven mad genes are unlinked.

Photomorphogenesis inPhycomyces: Fluence-response curves and action spectra

Blue light regulates vegetative reproduction inPhycomyces blakesleeanus Bgff. by inhibiting the development of microphores and stimulating that of macrophores. Fluence-response curves were obtained at twelve different wavelengths. Each response exhibits a two-step (“biphasic”) dependence on fluence, as if it resulted from the addition of two separate components with different thresholds, midpoints, and amplitudes. The absolute threshold is close to 10 photons·μm2. The threshold fluence of the low-intensity component is about 104 times smaller than that of the high-intensity component. The action spectra for each of the two components of the two responses share general similarities, but exhibit significant differences that might be taken to favour four separate photosystems. Additional complexity is indicated by the wavelength dependence of the saturation levels.

Mutants ofPhycomyces with enhanced tropisms

Abstract Seven mutants of Phycomyces which exhibit phototropism at high intensity of blue light (10 W/m 2 ) where the wild-type strain is unresponsive have been isolated. These mutants have the same absolute threshold for phototropism as wild type (10 −9 W/m 2 ). In comparison to wild type in the region just above this threshold, the mutants respond more strongly to light than to gravity, as determined by photogeotropic equilibrium experiments. The kinetics of phototropism, avoidance, and geotropism are also enhanced in the mutants. Therefore these mutants are designated phenotypically by the term “hypertropic.” The phenotype of these mutants is in many ways opposite to that of the so-called “stiff” mutants, which have slow tropisms. The rapid bending rates of the hypertropic mutants may be associated in part with the smaller diameter of their sporangiophores, but not according to a simple proportional relationship between phototropic bending rate and inverse diameter that applies for wild type and the hypergeotropic mutant C5. For photophorogenesis, which is mediated by the mycelium, one hypertropic mutant studied responds similarly to wild type. The pleiotropic character of these mutants suggests that they are affected near the output of the common sensory transduction pathway for these responses.

Photogeotropism inPhycomyces double mutants

Abstract Phycomyces sporangiophores exposed to continuous unilateral illumination reach an angle determined by the competition between phototropism and geotropism. This photogeotropism angle provides a quantitative assay for phototropic sensitivity as a function of light intensity. Seven unlinked genes have been associated with the sensory transduction chain for phototropism. A complete family of single and double mutant strains are available for a standard set of alleles for these genes. Photogeotropism measurements have been carried out comparatively on these strains as well as wild-type and one triple mutant affected in genes associated with early steps in the sensory pathway. These measurements were recorded over a millionfold range of light intensity. The results reveal a variety of reductions in sensitivity and responsiveness for the double mutants. Some of the double mutants, as well as the triple mutant, are extremely insensitive to light, but at the highest intensities none appear to be totally blind. The results are examined in the framework of two models for photogeotropism.

FOURIER ANALYSIS OF ANGULAR DISTRIBUTIONS FOR MOTILE MICROORGANISMS

The method of Fourier transformation is used to analyze histograms of the angular distribution of organisms moving with respect to a stimulus, in this case the light direction. In the Fourier spectra, the components with the highest amplitudes indicate the modality of orientation, i.e. the number of preferred directions, and the corresponding phase values indicate the deviation from the light direction. In the frequency domain, those components with high amplitude and/or those with low frequencies (low‐pass filtering) can be selected and used to reconstruct a smoothed histogram by means of an inverse Fourier transform. This method reduces the noise level and reveals the prominent features of histograms. Examples of unimodal, bimodal and multimodal distributions are shown in their original and smoothed form for the flagellate Euglena gracilis, and for amoebae and pseudoplasmodia or the slime mold Dictyostelium discoideum.

Video data acquisition for movement responses in individual organisms

Abstract— We describe a simple approach for the semiautomatic acquisition of data from video recordings of movement responses of microorganisms. For a sessile microorganism like Phycomyces blakesleeanus, we describe an angle transducer consisting of a precision rolary potentiometer and a transparent ruler. This device is placed in front of a video monitor during playback from a time‐lapse video recorder. The experimenter maintains the ruler parallel to the image of the upper region of the bending sporangiophore; a voltage proportional to the bending angle is directed to a strip‐chart recorder. The chart provides a continuous and precise graph of the bending angle as a function of time, so that the latency, bending rate, and other parameters may be readily measured. For motile microorganisms, such as Euglena gracilis, paths from video recordings are traced onto acetate sheets during playback. A rotary and a linear potentiometer convert the angle and length of successive path elements into analog voltages which are transmitted by analog‐to‐digital converters to a microcomputer for subsequent statistical and mathematical treatment. The general approaches presented here should be applicable to the study of movement responses of many types of organisms.

MRI/PET nonrigid breast-image registration using skin fiducial markers.

We propose a finite-element method (FEM) deformable breast model that does not require elastic breast data for nonrigid PET/MRI breast image registration. The model is applicable only if the stress conditions in the imaged breast are virtually the same in PET and MRI. Under these conditions, the observed intermodality displacements are solely due the imaging/reconstruction process. Similar stress conditions are assured by use of an MRI breast-antenna replica for breast support during PET, and use of the same positioning. The tetrahedral volume and triangular surface elements are used to construct the FEM mesh from the MRI image. Our model requires a number of fiducial skin markers (FSM) visible in PET and MRI. The displacement vectors of FSMs are measured followed by the dense displacement field estimation by first distributing the displacement, vectors linearly over the breast surface and then distributing them throughout the volume. Finally, the floating MRI image is warped to a fixed PET image, by using an appropriate shape function in the interpolation from mesh nodes to voxels. We tested our model on an elastic breast phantom with simulated internal lesions and on a small number of patients imaged, with FMS using PET and MRI. Using simulated lesions (in phantom) and real lesions (in patients) visible in both PET and MRI, we established that the target registration error (TRE) is below two pet voxels.