BAS1: A gene regulating brassinosteroid levels and light responsiveness in Arabidopsis

The Arabidopsis bas1-D mutation suppresses the long hypocotyl phenotype caused by mutations in the photoreceptor phytochrome B (phyB). The adult phenotype of bas1-D phyB-4 double mutants mimics that of brassinosteroid biosynthetic and response mutants. bas1-D phyB-4 has reduced levels of brassinosteroids and accumulates 26-hydroxybrassinolide in feeding experiments. The basis for the mutant phenotype is the enhanced expression of a cytochrome P450 (CYP72B1). bas1-D suppresses a phyB-null allele, but not a phyA-null mutation, and partially suppresses a cryptochrome-null mutation. Seedlings with reduced BAS1 expression are hyperresponsive to brassinosteroids in a light-dependent manner and display reduced sensitivity to light under a variety of conditions. Thus, BAS1 represents one of the control points between multiple photoreceptor systems and brassinosteroid signal transduction.

Virulence and Functions of Myosin II Are Inhibited by Overexpression of Light Meromyosin in Entamoeba histolytica

Several changes in cell morphology take place during the capping of surface receptors in Entamoeba histolytica. The amoebae develop the uroid, an appendage formed by membrane invaginations, which accumulates ligand–receptor complexes resulting from the capping process. Membrane shedding is particularly active in the uroid region and leads to the elimination of accumulated ligands. This appendage has been postulated to participate in parasitic defense mechanisms against the host immune response, because it eliminates complement and specific antibodies bound to the amoeba surface. The involvement of myosin II in the capping process of surface receptors has been suggested by experiments showing that drugs that affect myosin II heavy-chain phosphorylation prevent this activity. To understand the role of this mechanoenzyme in surface receptor capping, a myosin II dominant negative strain was constructed. This mutant is the first genetically engineered cytoskeleton-deficient strain of E. histolytica. It was obtained by overexpressing the light meromyosin domain, which is essential for myosin II filament formation. E. histolytica overexpressing light meromyosin domain displayed a myosin II null phenotype characterized by abnormal movement, failure to form the uroid, and failure to undergo the capping process after treatment with concanavalin A. In addition, the amoebic cytotoxic capacities of the transfectants on human colon cells was dramatically reduced, indicating a role for cytoskeleton in parasite pathogenicity.

Multiple pathways for ultrafast transduction of light energy in the photosynthetic reaction center of Rhodobacter sphaeroides

A pathway of electron transfer is described that operates in the wild-type reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides. The pathway does not involve the excited state of the special pair dimer of bacteriochlorophylls (P*), but instead is driven by the excited state of the monomeric bacteriochlorophyll (BA*) present in the active branch of pigments along which electron transfer occurs. Pump-probe experiments were performed at 77 K on membrane-bound RCs by using different excitation wavelengths, to investigate the formation of the charge separated state P+HA−. In experiments in which P or BA was selectively excited at 880 nm or 796 nm, respectively, the formation of P+HA− was associated with similar time constants of 1.5 ps and 1.7 ps. However, the spectral changes associated with the two time constants are very different. Global analysis of the transient spectra shows that a mixture of P+BA− and P* is formed in parallel from BA* on a subpicosecond time scale. In contrast, excitation of the inactive branch monomeric bacteriochlorophyll (BB) and the high exciton component of P (P+) resulted in electron transfer only after relaxation to P*. The multiple pathways for primary electron transfer in the bacterial RC are discussed with regard to the mechanism of charge separation in the RC of photosystem II from higher plants.

Vitamin B2-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals

In mammals the retina contains photoactive molecules responsible for both vision and circadian photoresponse systems. Opsins, which are located in rods and cones, are the pigments for vision but it is not known whether they play a role in circadian regulation. A subset of retinal ganglion cells with direct projections to the suprachiasmatic nucleus (SCN) are at the origin of the retinohypothalamic tract that transmits the light signal to the master circadian clock in the SCN. However, the ganglion cells are not known to contain rhodopsin or other opsins that may function as photoreceptors. We have found that the two blue-light photoreceptors, cryptochromes 1 and 2 (CRY1 and CRY2), recently discovered in mammals are specifically expressed in the ganglion cell and inner nuclear layers of the mouse retina. In addition, CRY1 is expressed at high level in the SCN and oscillates in this tissue in a circadian manner. These data, in conjunction with the established role of CRY2 in photoperiodism in plants, lead us to propose that mammals have a vitamin A-based photopigment (opsin) for vision and a vitamin B2-based pigment (cryptochrome) for entrainment of the circadian clock.

Effects of elevated atmospheric CO2 concentration on leaf dark respiration of Xanthium strumarium in light and in darkness

Leaf dark respiration (R) is an important component of plant carbon balance, but the effects of rising atmospheric CO2 on leaf R during illumination are largely unknown. We studied the effects of elevated CO2 on leaf R in light (RL) and in darkness (RD) in Xanthium strumarium at different developmental stages. Leaf RL was estimated by using the Kok method, whereas leaf RD was measured as the rate of CO2 efflux at zero light. Leaf RL and RD were significantly higher at elevated than at ambient CO2 throughout the growing period. Elevated CO2 increased the ratio of leaf RL to net photosynthesis at saturated light (Amax) when plants were young and also after flowering, but the ratio of leaf RD to Amax was unaffected by CO2 levels. Leaf RN was significantly higher at the beginning but significantly lower at the end of the growing period in elevated CO2-grown plants. The ratio of leaf RL to RD was used to estimate the effect of light on leaf R during the day. We found that light inhibited leaf R at both CO2 concentrations but to a lesser degree for elevated (17–24%) than for ambient (29–35%) CO2-grown plants, presumably because elevated CO2-grown plants had a higher demand for energy and carbon skeletons than ambient CO2-grown plants in light. Our results suggest that using the CO2 efflux rate, determined by shading leaves during the day, as a measure for leaf R is likely to underestimate carbon loss from elevated CO2-grown plants.

Basolateral membrane Na+/H+ exchange enhances HCO3- absorption in rat medullary thick ascending limb: evidence for functional coupling between basolateral and apical membrane Na+/H+ exchangers.

The role of basolateral membrane Na+/H+ exchange in transepithelial HCO3- absorption (JHCO3) was examined in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. In Na(+)-free solutions, addition of Na+ to the bath resulted in a rapid, amiloride-sensitive increase in intracellular pH. In MTALs perfused and bathed with solutions containing 146 mM Na+ and 25 mM HCO3-, bath addition of amiloride (1 mM) or 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 50 microM) reversibly inhibited JHCO3 by 50%. Evidence that the inhibition of JHCO3 by bath amiloride was the result of inhibition of Na+/H+ exchange included the following: (i) the IC50 for amiloride was 5-10 microM, (ii) EIPA was a 50-fold more potent inhibitor than amiloride, (iii) the inhibition by bath amiloride was Na+ dependent, and (iv) significant inhibition was observed with EIPA as low as 0.1 microM. Fifty micromolar amiloride or 1 microM EIPA inhibited JHCO3 by 35% when added to the bath but had no effect when added to the tubule lumen, indicating that addition of amiloride to the bath did not directly inhibit apical membrane Na+/H+ exchange. In experiments in which apical Na+/H+ exchange was assessed from the initial rate of cell acidification following luminal EIPA addition, bath EIPA secondarily inhibited apical Na+/H+ exchange activity by 46%. These results demonstrate basolateral membrane Na+/H+ exchange enhances transepithelial HCO3- absorption in the MTAL. This effect appears to be the result of cross-talk in which an increase in basolateral membrane Na+/H+ exchange activity secondarily increases apical membrane Na+/H+ exchange activity.

Roles of heavy and light chains in IgM polymerization.

IgM antibodies are secreted as multisubunit polymers that consist of as many as three discrete polypeptides: mu heavy chains, light (L) chains, and joining (J) chains. We wished to determine whether L chains that are required to confer secretory competence on immunoglobulin molecules must be present for IgM to polymerize--that is, for intersubunit disulfide bonds to form between mu chains. Using a L-chain-loss variant of an IgM-secreting hybridoma, we demonstrated that mu chains were efficiently polymerized independent of L chains, in a manner similar to that observed for conventional microL complexes, and that the mu polymers incorporated J chain. These mu polymers were not secreted but remained associated with the endoplasmic reticulum-resident chaperone BiP (GRP78). This finding is consistent with the endoplasmic reticulum being the subcellular site of IgM polymerization. We conclude that mu chain alone has the potential to direct the polymerization of secreted IgM, a process necessary but not sufficient for IgM to attain secretory competence.

Electrogenic light reactions in photosystem I: resolution of electron-transfer rates between the iron-sulfur centers.

Flash-induced voltage changes (electrogenic events) in photosystem I particles from spinach, oriented in a phospholipid layer, have been studied at room temperature on a time scale ranging from 1 micros to several seconds. A phospholipid layer containing photosystem I particles was adsorbed to a Teflon film separating two aqueous compartments. Voltage changes were measured across electrodes immersed in the compartments. In the absence of added electron donors and acceptors, a multiphasic voltage increase, associated with charge separation, was followed by a decrease, associated with charge recombination. Several kinetic phases were resolved: a rapid (<1 micros) increase, ascribed to electron transfer from the primary electron donor P700 to the iron-sulfur electron acceptor FB, was followed by a slower, biphasic increase with time constants of 30 and 200 micros. The 30-micros phase is assigned to electron transfer from FB to the iron-sulfur center FA. The voltage decrease had a time constant of 90 ms, ascribed to charge recombination from FA to P700. Upon chemical prereduction of FA and FB the 30- and 200-micros phases disappeared and the decay time constant was accelerated to 330 micros, assigned to charge recombination from the phylloquinone electron acceptor (A1) or the iron-sulfur center FX to P700.

On the absorbance changes in the photocycle of the photoactive yellow protein: A quantum-chemical analysis

Spectral changes in the photocycle of the photoactive yellow protein (PYP) are investigated by using ab initio multiconfigurational second-order perturbation theory at the available structures experimentally determined. Using the dark ground-state crystal structure [Genick, U. K., Soltis, S. M., Kuhn, P., Canestrelli, I. L. & Getzoff, E. D. (1998) Nature (London) 392, 206–209], the ππ* transition to the lowest excited state is related to the typical blue-light absorption observed at 446 nm. The different nature of the second excited state (nπ*) is consistent with the alternative route detected at 395-nm excitation. The results suggest the low-temperature photoproduct PYPHL as the most plausible candidate for the assignment of the cryogenically trapped early intermediate (Genick et al.). We cannot establish, however, a successful correspondence between the theoretical spectrum for the nanosecond time-resolved x-ray structure [Perman, B., Šrajer, V., Ren, Z., Teng, T., Pradervand, C., et al. (1998) Science 279, 1946–1950] and any of the spectroscopic photoproducts known up to date. It is fully confirmed that the colorless light-activated intermediate recorded by millisecond time-resolved crystallography [Genick, U. K., Borgstahl, G. E. O., Ng, K., Ren, Z., Pradervand, C., et al. (1997) Science 275, 1471–1475] is protonated, nicely matching the spectroscopic features of the photoproduct PYPM. The overall contribution demonstrates that a combined analysis of high-level theoretical results and experimental data can be of great value to perform assignments of detected intermediates in a photocycle.

Rethinking the role of phosducin: Light-regulated binding of phosducin to 14-3-3 in rod inner segments

Phosducin (Pd), a small protein found abundantly in photoreceptors, is widely assumed to regulate light sensitivity in the rod outer segment through interaction with the heterotrimeric G protein transducin. But, based on histochemistry and Western blot analysis, Pd is found almost entirely in the inner segment in both light and dark, most abundantly near the rod synapse. We report a second small protein, 14-3-3, in the rod with a similar distribution. By immunoprecipitation, phospho-Pd is found to interact with 14-3-3 in material from dark-adapted retina, and this interaction is markedly diminished by light, which dephosphorylates Pd. Conversely, unphosphorylated Pd binds to inner segment G protein(s) in the light. From these results and reported functions of 14-3-3, we have constructed a hypothesis for the regulation of light sensitivity at the level of rod synapse. By dissociating the Pd/14-3-3 complex, light enables both proteins to function in this role.

Architecture and mechanism of the light-harvesting apparatus of purple bacteria

Photosynthetic organisms fuel their metabolism with light energy and have developed for this purpose an efficient apparatus for harvesting sunlight. The atomic structure of the apparatus, as it evolved in purple bacteria, has been constructed through a combination of x-ray crystallography, electron microscopy, and modeling. The detailed structure and overall architecture reveals a hierarchical aggregate of pigments that utilizes, as shown through femtosecond spectroscopy and quantum physics, elegant and efficient mechanisms for primary light absorption and transfer of electronic excitation toward the photosynthetic reaction center.

An anion channel in Arabidopsis hypocotyls activated by blue light.

A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition.

Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light.

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery for signal, transduction required by the CRY1 protein is conserved among different plant species. Furthermore, the level of these photoresponses is seen to be determined by the cellular concentration of this photoreceptor.

Two routes of chlorophyllide synthesis that are differentially regulated by light in barley (Hordeum vulgare L.).

NADPH-protochlorophyllide oxidoreductase (POR; EC 1.6.99.1) catalyzes the only known light-dependent step in chlorophyll synthesis of higher plants, the reduction of protochlorophyllide (Pchlide) to chlorophyllide. In barley, two distinct immunoreactive POR proteins were identified. In contrast to the light-sensitive POR enzyme studied thus far (POR-A), levels of the second POR protein remained constant in seedlings during the transition from dark growth to the light and in green plants. The existence of a second POR-related protein was verified by isolating and sequencing cDNAs that encode a second POR polypeptide (POR-B) with an amino acid sequence identity of 75% to the POR-A. In the presence of NADPH and Pchlide, the in vitro-synthesized POR-A and POR-B proteins could be reconstituted to ternary enzymatically active complexes that reduced Pchlide to chlorophyllide only after illumination. Even though the in vitro activities of the two enzymes were similar, the expression of their genes during the light-induced transformation of etiolated to green seedlings was distinct. While the POR-A mRNA rapidly declined during illumination of dark-grown seedlings and soon disappeared, POR-B mRNA remained at an approximately constant level in dark-grown and green seedlings. Thus these results suggest that chlorophyll synthesis is controlled by two light-dependent POR enzymes, one that is active only transiently in etiolated seedlings at the beginning of illumination and the other that also operates in green plants.

pH gating of ROMK (Kir1.1) channels: Control by an Arg-Lys-Arg triad disrupted in antenatal Bartter syndrome

Inward-rectifier K+ channels of the ROMK (Kir1.1) subtype are responsible for K+ secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range. Here we show that a lysine residue close to TM1, identified previously as a structural element required for pH-induced gating, is protonated at neutral pH and that this protonation drives pH gating in ROMK and other Kir channels. Such anomalous titration of this lysine residue (Lys-80 in Kir1.1) is accomplished by the tertiary structure of the Kir protein: two arginines in the distant N and C termini of the same subunit (Arg-41 and Arg-311 in Kir1.1) are located in close spatial proximity to the lysine allowing for electrostatic interactions that shift its pKa into the neutral pH range. Structural disturbance of this triad as a result from a number of point mutations found in patients with antenatal Bartter syndrome shifts the pKa of the lysine residue off the neutral pH range and results in channels permanently inactivated under physiological conditions. Thus, the results provide molecular understanding for normal pH gating of Kir channels as well as for the channel defects found in patients with antenatal Bartter syndrome.