Photocontrol of Hypocotyl Elongation in Light-Grown Cucumis sativus L. : Responses to Phytochrome Photostationary State and Fluence Rate

The effects of the calculated photostationary state of phytochrome (_c) and the photon fluence rate on the elongation growth of the hypocotyl of light-grown seedlings of Cucumis sativus L. are examined. Two threshold responses to _c are found at values of 0.06 and 0.43. At _c = 0.06, there is no response at any fluence rate. In the _c range 0.1 to 0.43, elongation growth does not respond to changes in _c. Above the second threshold (_c = 0.43), there is a strong response to changes in _c. At all values of _c at and above 0.1, there is a response to fluence rate. A linear relationship can be demonstrated between a factor comprised of the logarithm of phytochrome cycling rate (a fluence-rate-dependent process) and _c, and the growth response.     

Effect of Monoclonal Antibodies on the in Vitro PFR Dark Reversion of Pea Phytochrome

Extraction as P_FR and immunoaffinity chromatography yieldeda pea phytochrome sample with polypeptide size of 121 kdalton,the same as in a crude extract which was immediately heatedin SDS. A difference spectrum was almost the same as that observedin etiolated pea epicotyls except that A₆₆₆/A₇₃₀ of 1.20 wassignificantly larger. At 10C dark reversion from P_FR occurred,with the decrease in A₇₂₈ being almost equal to the increasein A₆₆₇. The kinetics could be resolved into three first-ordercomponents, the major, slow component accounting for more than90% of the absorbance changes. In the presence of monoclonalanti-pea phytochrome antibodies mAP-1, 3 or 5, which bind awayfrom the chromophore, and mAP-7, which binds near the chromophore,the rate of the major component was reduced at either one orboth wavelengths. None of these antibodies affected the absorptionspectra of phytochrome. In the presence of mAP-9, which is suggestedto bind near the amino-terminus, the absorption at the red-light-inducedphotostationary state was reduced and the rate of dark reversionwas increased, resembling partially degraded phytochrome of114 kdalton, but with no evidence of proteolysis. ¹ Permanent address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan.     

Effect of Neutral Salts on Spectral Characteristics of Undegraded Phytochrome Partially Purified from Etiolated Pea Shoots

Spectral characteristics of partially purified undegraded peaphytochrome were investigated in different ionic conditions.At the red-light-induced photostationary state in low ionicstrength buffer phytochrome had reduced absorbance in its far-redpeak as reported previously. Elevation of the ionic strengthof the buffer reversibly increased the absorbance in the far-redregion at the photostationary state. It was found that the effectof increase of ionic strength was strengthened secondarily bychaotropicity of salts. It was confirmed that phytochrome preparations of low ionicstrength contained a photosensitive component(s) other thanthe red-light-absorbing form (P_R) and farred- light-absorbingform (P_FR) during photochemical transformation, as well as duringthe first several min in the dark after phototransformation.At high ionic strength, phytochrome became a two-component systemcomposed of only P_R and P_FR at the redlight-induced photostationarystate though a significant accumulation of another component(s)occurred during phototransformations. Increasing ionic strengthalso enhanced A723 of phytochrome at the red-light-induced photostationarystate. The effect could result from either an increased molefraction of P_FR at the photostationary state induced by redlight, or a change in the extinction coefficients of P_FR. ¹ Present address: Division of Biological Regulation, NationalInstitute for Basic Biology, Myodaijicho, Okazaki 444, Japan (Received March 18, 1981; Accepted August 3, 1981)     

A Novel Calibration-Minimum Method for Prediction of Mole Fraction in Non-Ideal Mixture

This article proposes a novel concentration prediction model that requires little training data and is useful for rapid process understanding. Process analytical technology is currently popular, especially in the pharmaceutical industry, for enhancement of process understanding and process control. A calibration-free method, iterative optimization technology (IOT), was proposed to predict pure component concentrations, because calibration methods such as partial least squares, require a large number of training samples, leading to high costs. However, IOT cannot be applied to concentration prediction in non-ideal mixtures because its basic equation is derived from the Beer–Lambert law, which cannot be applied to non-ideal mixtures. We proposed a novel method that realizes prediction of pure component concentrations in mixtures from a small number of training samples, assuming that spectral changes arising from molecular interactions can be expressed as a function of concentration. The proposed method is named IOT with virtual molecular interaction spectra (IOT-VIS) because the method takes spectral change as a virtual spectrum x _nonlin,i into account. It was confirmed through the two case studies that the predictive accuracy of IOT-VIS was the highest among existing IOT methods.     

Differences between the Surface Properties of the PR and PFR Forms of Native Pea Phytochrome, and Their Application to a Simplified Procedure for Purification of the Phytochrome

The ability of phytochrome from etiolated pea shoots (Pisumsativum L. cv. Alaska) to bind to various chromatographic adsorbentsand its mobility during non-denaturing electrophoresis wereexamined with phytochrome in either the red light-absorbingform (PR) or the far-red light-absorbing form (P_FR). Preferentialbinding of P_FR to modified hydrophilic polyvinyl resins, suchas butyl Toyopearl, phenyl Toyopearl, Blue Toyopearl (CibacronBlue F3G-A conjugated) and Red Toyopearl (Procion Red HE-3Bconjugated), was observed. A simplified method for purificationof native phytochrome was developed based on the propertiesof P_R and P_FR. P_FR bound preferentially to the hydrophobic adsorbents,to indicate that the surface of P_FR is more hydrophobic thanthat of P_R. A difference in net surface charges between P_R andP_FR was detected by an analysis based on the different mobilitiesof the two forms during non-denaturing polyacrylamide gel electrophoresisin gels prepared with various concentrations of polyacrylamide.The apparent molecular weights of P_R and P_FR, estimated fromthe analysis, were 378 and 419 kilodaltons, respectively. Thedifference suggests that a significant change in molecular shapeoccurs during the photoconversion. The differences in surfaceproperties of P_R and P_FR are discussed. (Received April 20, 1991; Accepted August 26, 1991)     

Reinterpreting the Anomalous Mole Fraction Effect: The Ryanodine Receptor Case Study

The origin of the anomalous mole fraction effect (AMFE) in calcium channels is explored with a model of the ryanodine receptor. This model predicted and experiments verified new AMFEs in the cardiac isoform. In mole fraction experiments, conductance is measured in mixtures of ion species X and Y as their relative amounts (mole fractions) vary. This curve can have a minimum (an AMFE). The traditional interpretation of the AMFE is that multiple interacting ions move through the pore in a single file. Mole fraction curves without minima (no AMFEs) are generally interpreted as X displacing Y from the pore in a proportion larger than its bath mole fraction (preferential selectivity). We find that the AMFE is also caused by preferential selectivity of X over Y, if X and Y have similar conductances. This is a prediction applicable to any channel and provides a fundamentally different explanation of the AMFE that does not require single filing or multiple occupancy: preferential selectivity causes the resistances to current flow in the baths, channel vestibules, and selectivity filter to change differently with mole fraction, and produce the AMFE.     

Dichroic Orientation of Phytochrome Intermediates in the Pathway from PR to PFR as Analyzed by Double Laser Flash Irradiations in Polarotropism of Adiantum Protonemata

The polarotropic response in protonemata of the fern Adiantumis regulated by phytochrome (Kadota et al. 1984); P_R and P_FRhave been shown to be dichroically oriented parallel and normalto the cell surface, respectively (Kadota et al. 1982). Thischange in the dichroic orientation of phytochrome during photoconversionwas analyzed by a newly-built, polarization plane-rotatabledouble laser flash irradiator. A polarotropic response was effectivelyinduced with a flash of polarized red (640 nm) light (6xl0^–7s) having the vibration plane of the electrical vector parallelto the protonemal cell axis. When a flash of polarized far-red(710 nm) light (6xl0^–7s) was given 30 sec after the redflash, the red flash-induced response was reversed by a far-redflash vibrating normal to the cell axis but not by one vibratingparallel. However, when given 2 µs or 2 ms after the redflash, the polarotropic response was not reversed by a polarizedfar-red flash vibrating normal to the cell axis but was reversedby a parallel-vibrating flash. These results suggest that theorientation of phototransformation intermediates existing 2µs or 2 ms after a red flash is still parallel to thecell surface, and that the change in the orientation of phytochromemolecules occurs between 2 ms and 30 s after the red flash. (Received February 3, 1986; Accepted April 23, 1986)     

Phytochrome: A Re-examination of the Quaternary Structure

Highly purified phytochrome samples from rye (Secale Cereale cv. Cougar) were fractionated by ultracentrifugation in isokinetic sucrose density gradients. Three protein species were separated with estimated sedimentation coefficients of 6.5S, 8.0S, and 11.5S. The 6.5S and 8.0S forms contained photoreversible phytochrome and produced a single subunit of 120,000 molecular weight upon reduction and electrophoresis in the presence of sodium dodecyl sulfate. The 11.5S species contained no detectable phytochrome. Reduction and electrophoresis of the 11.5S species in the presence of sodium dodecyl sulfate produced a major polypeptide of 32,000 molecular weight and a minor polypeptide of 48,000 molecular weight. The square tetrameric structures, observed by electron microscopy and previously thought to be phytochrome molecules, were found to be due to the presence of this 11.5S species in phytochrome preparations.     

Different Phototransduction Kinetics of Phytochrome A and Phytochrome B in Arabidopsis thaliana

The kinetics of phototransduction of phytochrome A (phyA) and phytochrome B (phyB) were compared in etiolated Arabidopsis thaliana seedlings. The responses of hypocotyl growth, cotyledon unfolding, and expression of a light-harvesting chlorophyll a/b-binding protein of the photosystem II gene promoter fused to the coding region of β-glucuronidase (used as a reporter enzyme) were mediated by phyA under continuous far-red light (FR) and by phyB under continuous red light (R). The seedlings were exposed hourly either to n min of FR followed by 60 minus n min in darkness or to n min of R, 3 min of FR (to back-convert phyB to its inactive form), and 57 minus n min of darkness. For the three processes investigated here, the kinetics of phototransduction of phyB were faster than that of phyA. For instance, 15 min R h⁻¹ (terminated with a FR pulse) were almost as effective as continuous R, whereas 15 min of FR h⁻¹ caused less than 30% of the effect of continuous FR. This difference is interpreted in terms of divergence of signal transduction pathways downstream from phyA and phyB.     

Phytochrome B Enhances Photosynthesis at the Expense of Water-Use Efficiency in Arabidopsis

In open places, plants are exposed to higher fluence rates of photosynthetically active radiation and to higher red to far-red ratios than under the shade of neighbor plants. High fluence rates are known to increase stomata density. Here we show that high, compared to low, red to far-red ratios also increase stomata density in Arabidopsis (Arabidopsis thaliana). High red to far-red ratios increase the proportion of phytochrome B (phyB) in its active form and the phyB mutant exhibited a constitutively low stomata density. phyB increased the stomata index (the ratio between stomata and epidermal cells number) and the level of anphistomy (by increasing stomata density more intensively in the adaxial than in the abaxial face). phyB promoted the expression of FAMA and TOO MANY MOUTHS genes involved in the regulation of stomata development in young leaves. Increased stomata density resulted in increased transpiration per unit leaf area. However, phyB promoted photosynthesis rates only at high fluence rates of photosynthetically active radiation. In accordance to these observations, phyB reduced long-term water-use efficiency estimated by the analysis of isotopic discrimination against ¹³CO₂. We propose a model where active phyB promotes stomata differentiation in open places, allowing plants to take advantage of the higher irradiances at the expense of a reduction of water-use efficiency, which is compensated by a reduced leaf area.Photosynthesis, transpiration, and transpiration efficiency, the ratio of carbon fixation to water loss, are key physiological traits considered by plant breeders when selecting productive and water-use efficient plants (Rebetzke et al., 2002; Richards, 2006; Passioura, 2007). Opening of the stomata allows the uptake of CO₂ necessary for photosynthesis but it simultaneously increases the loss of water and the potential deterioration of the water status. Plants are finely tuned to efficiently face this dilemma. Under low levels of photosynthetically active radiation (PAR), stomata open just enough to prevent the limitation of photosynthesis by CO₂ influx and the photochemical phase of photosynthesis is the limiting step. If PAR increases, allowing higher rates of photochemical reactions, which leads to more ATP and NADPH, stomatal conductance also increases to allow sufficient CO₂ to use these products in the Calvin cycle (Donahue et al., 1997; Yu et al., 2004). If instead of following this response coordinated to photosynthetic rates, stomata opened maximally in response to low PAR, more CO₂ than needed would be allowed to reach the chloroplast at the expense of unnecessary water loss.Canopy shade light is characterized not only by reduced PAR levels but also by a reduced proportion of red light (R) compared to far-red light (FR) caused by the selective absorption of visible light by photosynthetic pigments and the reflection and transmission of FR (Holmes and Smith, 1977a). This low R/FR ratio compared to unfiltered sunlight is perceived by phytochromes (Smith, 1982; Ballaré et al., 1987; Pigliucci and Schmitt, 1999), mainly phytochrome B (phyB; Yanovsky et al., 1995). In Arabidopsis (Arabidopsis thaliana), the high R/FR signals perceived by phyB decrease the length of the stem and petioles, cause a more prostrate position of the leaves, and promote branching and delay flowering, among other responses (Reed et al., 1993; Franklin and Whitelam, 2005).Transgenic plants of potato (Solanum tuberosum) expressing the PHYB gene of Arabidopsis show higher stomatal conductance, transpiration rates, and photosynthesis rates per unit leaf area than the wild type (Thiele et al., 1999; Boccalandro et al., 2003; Schittenhelm et al., 2004). Stomata density is unaffected, indicating that phyB enhances the aperture of the stomatal pore in these transgenic plants. Stomatal conductance is higher in Fuchsia magellanica plants exposed to R than to FR pulses at the end of the photoperiod (Aphalo et al., 1991). However, there are no general effects of R/FR treatments on the aperture of the stomatal pore. The stomata of Commelina communis (Roth-Bejerano, 1981) and of the orchid of the genus Paphiopedilum (Talbott et al., 2002) open in response to R and this effect is reversed by FR, indicating a control by phytochrome. Nevertheless, this FR reversal of the effect of R is absent in wild-type Arabidopsis (Talbott et al., 2003). In Phaseolus vulgaris, FR accelerates stomatal movements during dark to light (opening) and light to dark (closing) transitions and this effect is R reversible, but phytochrome status has no effects under constant conditions of light or darkness (Holmes and Klein, 1985). In the latter species, prolonged FR added to a white-light background promotes stomatal conductance but this effect cannot be ascribed to phytochrome (Holmes et al., 1986).In addition to this rapid adjustment of the CO₂ and water vapor fluxes to daily fluctuations in light levels via the regulation of the stomatal pore aperture, plants acclimate to the prevailing PAR conditions by changing stomatal density (number of stomata per unit area) and stomatal index (the ratio between the number of stomata in a given area and the total number of stomata and other epidermal cells in that same area). Stomatal density and stomatal index are higher in plants grown in full sunlight at high levels of PAR than in plants grown in shade (Willmer and Fricker, 1996; Lake et al., 2001; Thomas et al., 2004; Casson and Gray, 2008). Mature leaves sense the environment (light intensity and CO₂) and produce a systemic signal that regulates stomatal density and index in young leaves (Coupe et al., 2006). A change in CO₂ concentrations or PAR levels affects photosynthesis and therefore it was suggested that a metabolic compound associated to this process (i.e. a sugar) may regulate stomatal development (Coupe et al., 2006). However, there is no correlation between photosynthetic rate and stomatal index in poplar (Populus spp.; Miyazawa et al., 2006) and transgenic anti-small subunit of Rubisco tobacco (Nicotiana tabacum) plants, show reduced photosynthesis and normal responses of stomatal density and stomatal index to PAR, suggesting that other photoreceptors could be involved in this regulation (Baroli et al., 2008).Here we demonstrate that high, compared to low, R/FR ratios perceived by phyB increase stomata density, stomata index, and amphistomy in the leaves of Arabidopsis. This behavior results in an enhanced photosynthetic rate at high PAR at the expense of reduced water-use efficiency.     

A Re-evaluation of Soybean Leaf Photorespiration

Recalculations of soybean photorespiration indicate that mean rates are closer to 16.1 than 5.6 milligrams of CO₂ per square decimeter per hour as previously reported. Photorespiration of soybean thus amounts to at least a 30% carbon turnover of light-saturated photosynthesis. Photorespiration showed no significant relationship to net photosynthesis. Negative correlations were found between CO₂ efflux and stomatal resistance as well as between corrected photorespiration and residual intracellular resistance of the leaf to CO₂ uptake.     

Undegraded chromogranin A is present in serum and enters the endocytotic lysosomal pathway in kidney

Summary Analysis of human and bovine serum by immunoblotting revealed the presence of the proprotein chromogranin A. By the same method chromogranin A was also found in rat, bovine and human kidney. However this organ did not contain any chromogranin A mRNA arguing against a synthesis within this organ. By immun-electron microscopy chromogranin A immunoreactivity was found in proximal tubule cells of rat kidney. Positive immunostaining was present in small vesicles within and in close proximity to the brush border and closer to the nucleus in typical lysosomal structures. These results make it likely that chromogranin A from serum reaches kidney tubule cells by glomerular filtration and is taken up into the endocytotic lysosomal pathway.     

Undegraded chromogranin A is present in serum and enters the endocytotic lysosomal pathway in kidney.

Analysis of human and bovine serum by immunoblotting revealed the presence of the proprotein chromogranin A. By the same method chromogranin A was also found in rat, bovine and human kidney. However this organ did not contain any chromogranin A mRNA arguing against a synthesis within this organ. By immune-electron microscopy chromogranin A immunoreactivity was found in proximal tubule cells of rat kidney. Positive immunostaining was present in small vesicles within and in close proximity to the brush border and closer to the nucleus in typical lysosomal structures. These results make it likely that chromogranin A from serum reaches kidney tubule cells by glomerular filtration and is taken up into the endocytotic lysosomal pathway.     

A Polarity Probe for Monitoring Light-induced Structural Changes at the Entrance of the Chromophore Pocket in a Bacterial Phytochrome

Light-induced structural changes at the entrance of the chromophore pocket of Agp1 phytochrome were investigated by using a thiol-reactive fluorescein derivative that is covalently attached to the genuine chromophore binding site (Cys-20) and serves as a polarity probe. In the apoprotein, the absorption spectrum of bound fluorescein is red-shifted with respect to that of the free label suggesting that the probe enters the hydrophobic chromophore pocket. Assembly of this construct with the chromophores phycocyanobilin or biliverdin is associated with a blue-shift of the fluorescein absorption band indicating the displacement of the probe out of the pocket. The probe does not affect the photochromic and kinetic properties of the noncovalent bilin adducts. Upon photoconversion to P_fr, the probe spectrum undergoes again a bathochromic shift and a strong rise in CD indicating a more hydrophobic and asymmetric environment. We propose that the environmental changes of the probe reflect conformational changes at the entrance of the chromophore pocket and are indicative for rearrangements of the chromophore ring A. Flash photolysis measurements showed that the absorption changes of the probe are kinetically coupled to the formation of Meta-R_C and P_fr. In the biliverdin adduct, an additional component occurs that probably reflects a transition between two Meta-R_C substates. Analogous results to that of the noncovalent phycocyanobilin adduct were obtained with the mutant V249C in which probe and chromophore are covalently attached. The conformational changes of the chromophore are correlated to proton transfer to the protein surface.Phytochromes are red-light photoreceptors occurring in plants, bacteria, and fungi where they control important developmental processes (1–6). The discovery of microbial phytochromes from genome sequencing (7–9) provided new prospects for biochemical, spectroscopic and structural analyses of this light sensor family. Agp1 (AtBphP1)³ from the soil bacterium Agrobacterium tumefaciens is a typical member of the widespread family of proteobacterial phytochromes (10, 11) and is the subject of the present study.The domain arrangement of canonical phytochromes consists of an N-terminal photosensory domain, including PAS, GAF, and PHY domains and a C-terminal regulatory kinase domain (see, e.g. Ref. 3). Bacterial phytochromes lack the N-terminal extension, and the PAS module insertion of plant phytochromes (3). In most of the bacterial phytochromes, the C-terminal regulatory domain is a histidine kinase (4). These kinases form homodimers as functional units (12) where the subunits transphosphorylate each other (13). The cofactors are linear tetrapyrroles that are covalently attached via a thioether linkage (14) to the side chains of specific conserved cysteine residues. The native chromophore of plant phytochromes is phytochromobilin (PΦB) (14), some cyanobacterial phytochromes incorporate phycocyanobilin (PCB) (15, 16), and all other bacterial phytochromes bind biliverdin (BV) (10, 11). Whereas the chromophore binding site of the more reduced bilins PΦB and PCB is located in the GAF domain, the binding site of BV is close to the N terminus upstream of the PAS domain (4, 11). The two distinct binding sites apparently require a specific substituent at the C3 carbon of pyrrole ring A, either an ethylidene (PΦB and PCB) or a vinyl (BV) group, for covalent attachment of the bilin chromophore (4). The holophytochrome assembly that includes covalent attachment of the chromophore is an autocatalytic process implying an intrinsic bilin C-S lyase activity of the apophytochrome (17). Kinetic studies of the autoassembly in vitro showed that ligation of the chromophore is the ultimate step following incorporation in the binding pocket and internal protonation (18).Phytochromes display photochromicity involving two either thermally stable or long-lived states, P_r and P_fr (red and far-red absorbing forms), that can be reversibly converted by light of appropriate wavelengths. The P_r to P_fr photoconversion is initiated by a rapid Z/E isomerization of the C-D methine bridge of the bilin chromophore (19–22) leading within picoseconds to the formation of the Lumi-R intermediate (23, 24). The following thermal relaxations via Meta-R_A and Meta-R_C intermediates to P_fr proceed on the time scale of microseconds and milliseconds (25–28).Assembly of Agp1 with locked BV derivatives showed that the geometry of the C-D methine bridge is 15Z_anti in P_r and 15E_anti in P_fr (29) suggesting that this methine bridge remains in the anti conformation during photoconversion. The crystal structures of the chromophore binding domains of the bacteriophytochromes from Deinococcus radiodurans and Rhodopseudomonas palustris revealed that the BV chromophore adopts a 5Z_syn,10Z_syn,15Z_anti configuration/conformation in the P_r state (30–32). The 5Z_syn geometry of the A-B methine bridge in the P_r state was confirmed by assembly of Agp1 with the corresponding locked BV chromophore (33). Recently, heteronuclear NMR investigations and crystallographic studies on the complete photosensory domain of the cyanobacterial phytochrome Cph1 from Synechocystis showed that the PCB chromophore is also in the 5Z_syn,10Z_syn,15Z_anti geometry in P_r (34, 35).Because the locked 5Z_syn adduct of Agp1 did not show a P_fr-like photo-product, conformational changes of the A-B methine bridge in the thermal relaxation cascade have been predicted (33). Flash photolysis experiments with this adduct suggested that these changes occur in the Meta-R_A to Meta-R_C transition (36). The stereochemistry of the A-B methine bridge in the P_fr state and in the preceding intermediates could not be determined unambiguously yet. Recent studies with doubly locked chromophores suggest that the C5–C6 single bond undergoes a thermal rotation from syn to anti in the photoconversion of Agp1, whereas an additional Z/E isomerization around the C4C5 double bond (hula-twist mechanism) was postulated for Agp2 (37). However, the crystal structure of the photosensory domain of the bacteriophytochrome PaBphP in its P_fr-enriched dark-adapted state favors the 5Z_syn conformation of the BV chromophore (38). Structural changes of the A-B methine bridge were excluded for the PCB chromophore of Cph1 on the basis of heteronuclear NMR (34), whereas low temperature Fourier transform IR studies on plant phytochrome suggested an environmental change of the ring A carbonyl group and/or a twist of the A-B methine bridge (39).The mechanism by which the signal is transmitted from the bilin chromophore to the protein is still obscure. The recent three-dimensional structures of the complete photosensory domains of Cph1 (35) and PaBphP (38) reveal key interactions between GAF and PHY domains in the corresponding dark states reflecting P_r and P_fr, respectively. In view of the intrinsic differences between the two phytochromes, it is not trivial to differentiate which of the numerous structural differences arise from light-induced conformational changes and are thus potentially important for signal transmission. We note that many approaches to provide a clue on the mechanism of signal transmission from the bilin chromophore to its proximate environment imply that this process is exclusively coupled to the photo-isomerization localized at ring D and its environment and that the chromophore then remains a passive element in the thermal relaxation cascade. This point of view is supported by recent results from femtosecond stimulated Raman spectroscopy suggesting that the chromophore structures in Lumi-R and P_fr are very similar (24). On the other hand, size exclusion chromatography experiments demonstrated that the global conformational changes observed for the P_fr state of Agp1 WT are absent in constructs (locked 5Zs adduct and mutants D197A and H250A), where the formation of P_fr is inhibited but the primary photoreaction proceeds (33, 40). These results are difficult to explain in terms of an ultra-fast signal transmission from the chromophore to the surrounding residues in its pocket.Light-induced conformational changes at the surface of plant phytochrome were observed by using covalently attached labels that are sensitive to the polarity of the microenvironment (41, 42). Due to the accessibility of several binding sites (i.e. the sulfhydryl groups of cysteines) in these experiments, the labeling was unspecific preventing further assignment of the observed changes to particular regions of the protein. Time-resolved absorption measurements with a covalently attached fluorescein derivative showed that the changes occur in the Meta-R_C to P_fr transition (41). In the present work with Agp1 phytochrome, we take advantage of the highly reactive sulfhydryl group of Cys-20, the genuine binding site of the BV chromophore, to specifically attach a fluorescein derivative. We observed that this construct assembles with PCB and BV forming noncovalent photochromic adducts, spectrally and kinetically undisturbed by the fluorescein label. Upon photo-conversion, the absorption band of the label displays a bathochromic shift and increase in ellipticity suggesting that the label moves in a more hydrophobic and asymmetric environment in the P_fr state. The label thus serves as a polarity probe at the entrance of the binding pocket. We postulate that these polarity changes reflect conformational changes of the A-B methine of the bilin chromophore and/or the microenvironment of ring A at the entrance of the binding pocket. Time-resolved measurements reveal that the changes occur in the Meta-R_A to Meta-R_C and Meta-R_C to P_fr transitions. Analogous results were obtained with the V249C mutant of Agp1 in which both the fluorescein probe and the PCB chromophore are covalently attached.     

Identification of the Tryptophan Residue Located at the Substrate-binding Site of Rye Seed Chitinase-c

Chemical modifications of rye seed chitinase-c (RSC-c) with various reagents suggested the involvements of tryptophan and glutamic/aspartic acid residues in the activity. Of these, the modification of tryptophan residues with N-bromosuccinimide (NBS) was investigated in detail.

In the NBS-oxidation at pH 4.0, two of the six tryptophan residues in RSC-c were rapidly oxidized and the chitinase activity was almost completely lost. On the other hand, in the NBS-oxidation at pH 5.9, only one tryptophan residue was oxidized and the activity was greatly reduced. Analyses of the oxidized tryptophan-containing peptides from the tryptic and chymotryptic digests of the modified RSC-c showed that two tryptophan residues oxidized at pH 4.0 are Trp72 and Trp82, and that oxidized at pH 5.9 is Trp72.

The NBS-oxidation of Trp72 at pH 5.9 was protected by a tetramer of N-acetylglucosamine (NAG₄), a very slowly reactive substrate for RSC-c, and the activity was almost fully retained. In the presence of NAG4, RSC-c exhibited an UV -difference spectrum with maxima at 284 nm and 293 nm, attributed to the red shift of the tryptophan residue, as well as a small trough around 300 nm probably due to an alteration of the environment of the tryptophan residue. From these results, it was suggested that Trp72 is exposed on the surface of the RSC-c molecule and involved in the binding to substrate.     

A Comparison of Freezing Injury in Oat and Rye: Two Cereals at the Extremes of Freezing Tolerance

A detailed analysis of cold acclimation of a winter rye (Secale cereale L. cv Puma), a winter oat (Avena sativa L. cv Kanota), and a spring oat cultivar (Ogle) revealed that freezing injury of leaves of nonacclimated seedlings occurred at -2[deg]C in both the winter and spring cultivars of oat but did not occur in winter rye leaves until after freezing at -4[deg]C. The maximum freezing tolerance was attained in all cultivars after 4 weeks of cold acclimation, and the temperature at which 50% electrolyte leakage occurred decreased to -8[deg]C for spring oat, -10[deg]C for winter oat, and -21[deg]C for winter rye. In protoplasts isolated from leaves of nonacclimated spring oat, expansion-induced lysis was the predominant form of injury over the range of -2 to -4[deg]C. At temperatures lower than -4[deg]C, loss of osmotic responsiveness, which was associated with the formation of the hexagonal II phase in the plasma membrane and subtending lamellae, was the predominant form of injury. In protoplasts isolated from leaves of cold-acclimated oat, loss of osmotic responsiveness was the predominant form of injury at all injurious temperatures; however, the hexagonal II phase was not observed. Rather, injury was associated with the occurrence of localized deviations of the plasma membrane fracture plane to closely appressed lamellae, which we refer to as the "fracture-jump lesion." Although the freeze-induced lesions in the plasma membrane of protoplasts of spring oat were identical with those reported previously for protoplasts of winter rye, they occurred at significantly higher temperatures that correspond to the lethal freezing temperature.     

A Re-evaluation of the Nitrate Reductase Content of the Maize Root

The standard procedure for the in ritro extraction of nitrate reductase from the tip region (0-2 cm) of the primary root of the maize (Zea mays L.) seedling indicated an activity of the enzyme approximately 5-fold higher than that obtained with an in vivo assay. In more mature regions of the primary root the ratio of in vitro to in vivo activity was much lower and in older seedlings was less than unity. The mature root extracts had a more labile nitrate reductase and a higher level of an inactivating enzyme. The use of phenylmethylsulphonyl fluoride in the extraction medium gave only a partial protection of the nitrate reductase from the old root samples. Casein (3%) resulted in a greatly increased yield of nitrate reductase (36-fold with one sample) and a more constant in vitro-in vivo activity ratio for all root samples. With casein in the extraction medium, much higher levels of nitrate reductase were recovered from the mature root zone, and the root content of this enzyme was now shown to be quite a significant proportion of the total in the maize seedling. Casein was shown to inhibit the action of the inactivating enzyme on nitrate reductase. Evidence is also presented for a nitrate reductase inactivating enzyme in the maize scutella and leaf tissues and in the roots and shoots of pea seedlings.