Proton uptake by bacterial reaction centers: The protein complex responds in a similar manner to the reduction of either quinone acceptor

In bacterial photosynthetic reaction centers, the protonation events associated with the different reduction states of the two quinone molecules constitute intrinsic probes of both the electrostatic interactions and the different kinetic events occurring within the protein in response to the light-generated introduction of a charge. The kinetics and stoichiometries of proton uptake on formation of the primary semiquinone QA− and the secondary acceptor QB− after the first and second flashes have been measured, at pH 7.5, in reaction centers from genetically modified strains and from the wild type. The modified strains are mutated at the L212Glu and/or at the L213Asp sites near QB; some of them carry additional mutations distant from the quinone sites (M231Arg → Leu, M43Asn → Asp, M5Asn → Asp) that compensate for the loss of L213Asp. Our data show that the mutations perturb the response of the protein system to the formation of a semiquinone, how distant compensatory mutations can restore the normal response, and the activity of a tyrosine residue (M247Ala → Tyr) in increasing and accelerating proton uptake. The data demonstrate a direct correlation between the kinetic events of proton uptake that are observed with the formation of either QA− or QB−, suggesting that the same residues respond to the generation of either semiquinone species. Therefore, the efficiency of transferring the first proton to QB is evident from examination of the pattern of H+/QA− proton uptake. This delocalized response of the protein complex to the introduction of a charge is coordinated by an interactive network that links the Q− species, polarizable residues, and numerous water molecules that are located in this region of the reaction center structure. This could be a general property of transmembrane redox proteins that couple electron transfer to proton uptake/release reactions.

Water Deficit and Spatial Pattern of Leaf Development. Variability in Responses Can Be Simulated Using a Simple Model of Leaf Development1

We analyzed the effect of short-term water deficits at different periods of sunflower (Helianthus annuus L.) leaf development on the spatial and temporal patterns of tissue expansion and epidermal cell division. Six water-deficit periods were imposed with similar and constant values of soil water content, predawn leaf water potential and [ABA] in the xylem sap, and with negligible reduction of the rate of photosynthesis. Water deficit did not affect the duration of expansion and division. Regardless of their timing, deficits reduced relative expansion rate by 36% and relative cell division rate by 39% (cells blocked at the G0-G1 phase) in all positions within the leaf. However, reductions in final leaf area and cell number in a given zone of the leaf largely differed with the timing of deficit, with a maximum effect for earliest deficits. Individual cell area was only affected during the periods when division slowed down. These behaviors could be simulated in all leaf zones and for all timings by assuming that water deficit affects relative cell division rate and relative expansion rate independently, and that leaf development in each zone follows a stable three-phase pattern in which duration of each phase is stable if expressed in thermal time (C. Granier and F. Tardieu [1998b] Plant Cell Environ 21: 695–703).

Spatial and Temporal Analyses of Expansion and Cell Cycle in Sunflower Leaves1 : A Common Pattern of Development for All Zones of a Leaf and Different Leaves of a Plant

We have investigated the spatial distributions of expansion and cell cycle in sunflower (Helianthus annuus L.) leaves located at two positions on the stem, from leaf initiation to the end of expansion. Relative expansion rate (RER) was analyzed by following the deformation of a grid drawn on the lamina; relative division rate (RDR) and flow-cytometry data were obtained in four zones perpendicular to the midrib. Calculations for determining in situ durations of the cell cycle and of S-G2-M in the epidermis are proposed. Area and cell number of a given leaf zone increased exponentially during the first two-thirds of the development duration. RER and RDR were constant and similar in all zones of a leaf and in all studied leaves during this period. Reduction in RER occurred afterward with a tip-to-base gradient and lagged behind that of RDR by 4 to 5 d in all zones. After a long period of constancy, cell-cycle duration increased rapidly and simultaneously within a leaf zone, with cells blocked in the G0-G1 phase of the cycle. Cells that began their cycle after the end of the period with exponential increase in cell number could not finish it, suggesting that they abruptly lost their competence to cross a critical step of the cycle. Differences in area and in cell number among zones of a leaf and among leaves of a plant essentially depended on the timing of two events, cessation of exponential expansion and of exponential division.

The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern.

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by Kcat for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.