\u3ci\u3eIn vitro\u3c/i\u3e assembly of apophytochrome and apophytochrome deletion mutants expressed in yeast with phycocyanobilin

Recombinant pea type I phytochrome apoprotein expressed in yeast is shown to assemble in vitro with phycocyanobilin to produce a photoreversible phytochromelike adduct. As an initial investigation of the amino acid sequence requirements for chromophore incorporation, three phyA gene product deletion mutants were produced in yeast. Truncation of the N-terminal tail to residue 46 demonstrates that this region is not critical to bilin attachment, but a deletion mutant lacking 222 amino acids from the N terminus failed to yield holophytochrome in vitro, under the same conditions. A mutant comprising a deletion of the C terminus to residue 548 showed bilin incorporation and red/far-red photoreversibility, indicating that bilin-apophytochrome assembly still occurred even when the entire C-terminal domain was truncated

THE POLARIZED PHOSPHORESCENCE SPECTRA OF FLAVINS AT 77∘K77^{\circ}K.

This research was supported by the National Science Foundation Grant No. GB-8055.Author Institution: Department of Chemistry, Texas Technological CollegeSong, Pill-Soon Department of Chemistry Texas Technological College riboflavin and alloxazine were measured in ethylene glycol-water (2:1, v/v) at $77^{\circ}K$ in order to assign the lowest triplet state and $(n, \pi^{\ast})$ singlet state. It was found that the region of the spectrum around the 0-0 emission band is positively polarized with respect to the 350 nm singlet-singlet transition moment(s). Since it was also shown that the phosphorescence emission is negatively polarized with respect to the lowest singlet ($\pi, \pi^{\ast}$) and the phosphorescence excitation at 350 nm is positively polarized, the results are consistent with the triplet state being ($\pi, \pi^{\ast}$) and with the 350 nm band being of $(n, \pi^{\ast})$ character. The source of the triplet-singlet transition moments of flavins has also been studied by calculating one-center spin orbit matrix elements of the $^{1}(n, \pi^{\ast})-^{3}(\pi, \pi^{\ast}$) coupling

Structure and Function of the Phytochromes: Light Regulation of Plant Growth and Development

Light exerts two primary roles in plant growth and development. Plants acquire all biochemical energy required for growth and propagation solely from light energy via photosynthesis. In addition, light serves as a medium through which plants recognize environmental fluctuations, such as photoperiod and presence of neighboring animals and plants. Plants therefore constantly monitor the direction, intensity, duration, and wavelength of environmental light and integrate these light signals into the intrinsic regulatory programs to achieve an optimized growth in a given light condition. Although light regulates all aspects of plant growth and developmental aspects, the molecular mechanisms and signaling cascades involved have not been well established until recently. However, recent advances in genetic tools and plant transformation techniques greatly facilitated the elucidation of molecular events in plant photomorphogenesis. This mini-review summarizes the gist of recent findings in deetiolation and supresion of shade avoidance response as clasic examples of the phytochrome-mediated photomorphogenesis

MOLECULAR LUMINESCENCE SPECTRA OF COUMARINS AND FUROCOUMARINS AT 77∘K77^{\circ} K

Author Institution: Department of Chemistry, Texas Tech UniversityThe lowest excited singlet and triplet states of coumarins and furocoumarins (psoralens) have been assigned to the $(\pi,\pi^{*})$ type on the basis of luminescence and photoselection measurements in ethanol at $77^{\circ} K$, Surprisingly, coumarins and furoumarins without substituent at positions 3 and 4 of the pyrone moiety showed their 0-0 phosphorescence bands at essentially the same location regardless of size and substituent of these molecules. For example, the 0-0 bands are 457 (coumarin), 456, 450 (isopsoralen), 457 (8-methoxy-psoralen), and ca. 452 nm (4,5,8-trimethly psoralen). Polarizations of the 0-0 bands are negative or minimum, and out-of-plane vibration (possibly C-H) with maximum polarization is also present. 5-Methoxypsoralen shows its 0-0 band at 473 nm. Phosphorescence lifetimes of these compounds are found to be 0.5-1 sec. The above results are interpreted in terms of highly localized triplet states in the region of the pyrone double bond, and SCF molecular orbital data will be presented in support of the localized nature of the triplet states. Relative directions of the two transition moments $(\pi, \pi^{*})$ have also been determined from the lowest polarized fluorescence excitation spectra

Viscosity Dependence of Primary Photoprocesses of 124 Kilodalton Phytochrome

To characterize the nature of primary photoprocesses of phytochrome which serves as the red-far red reversible photoreceptor for photomorphogenesis in plants, viscosity dependence of the fluorescence lifetimes of phytochrome isolated from etiolated oat seedling (Avena sativa L.) has been investigated. The fluorescence decay of phytochrome exhibited approximately two components, one with lifetime in the range of 50-70 ps and another with 1.1-1.2 ns in phosphate buffer with or without 40-67% glycerol. However, relative amplitudes of these decay components were found to be strongly viscosity dependent. Thus, the longer decay component increased from 2-5% in phosphate buffer to ∼20% in 67% glycerol-phosphate buffer. These results have been interpreted in terms of primary reaction from the excited singlet state of phytochrome, yielding a photoreversible intermediate whose rate of formation and decay were apparently viscosity-dependent. Further, the viscosity dependence is consistent with the primary reaction involving conformational changes of the chromophore/its apoprotein environment

Protein phosphorylation in isolated nuclei from etiolated Avena seedlings Effects of red/far-red light and cholera toxin

AbstractWe have studied the phosphorylation/dephosphorylation of several nuclear proteins in isolated nuclei from etiolated Avena seedlings as a function of red/far-red light. The effect of stimulatory (ADP-ribosylation by cholera toxin) or inhibitory (GDPβS) conditions for GTP-binding proteins was also studied. Red or far-red light enhanced the phosphorylation level of 2 nuclear proteins with molecular masses of 75 and 60 kDa. The phosphorylation pattern was affected by the addition of cholera toxin or GDPβS to the isolated nuclei. At least 2 proteins with molecular masses of 24 and 75 kDa cross-reacted by Westerrs blot with GTP-binding protein antibodies