James A. Guikema

Rice cationic peroxidase accumulates in xylem vessels during incompatible interactions with Xanthomonas oryzae pv oryzae.

A cationic peroxidase, PO-C1 (molecular mass 42 kD, isoelectric point 8.6), which is induced in incompatible interactions between the vascular pathogen Xanthomonas oryzae pv oryzae and rice (Oryza sativa L.), was purified. Amino acid sequences from chemically cleaved fragments of PO-C1 exhibited a high percentage of identity with deduced sequences of peroxidases from rice, barley, and wheat. Polyclonal antibodies were raised to an 11-amino acid oligopeptide (POC1a) that was derived from a domain where the sequence of the cationic peroxidase diverged from other known peroxidases. The anti-POC1a antibodies reacted only with a protein of the same mobility as PO-C1 in extracellular and guttation fluids from plants undergoing incompatible responses collected at 24 h after infection. In the compatible responses, the antibodies did not detect PO-C1 until 48 h after infection. Immunoelectron microscopy was used to demonstrate that PO-C1 accumulated within the apoplast of mesophyll cells and within the cell walls and vessel lumen of xylem elements of plants undergoing incompatible interactions.

Identification of Surface-Exposed Domains on the Reducing Side of Photosystem I

Photosystem I (PSI) is a multisubunit enzyme that catalyzes the light-driven oxidation of plastocyanin or cytochrome c6 and the concomitant photoreduction of ferredoxin or flavodoxin. To identify the surface-exposed domains in PSI of the cyanobacterium Synechocystis sp. PCC 6803, we mapped the regions in PsaE, PsaD, and PsaF that are accessible to proteases and N-hydroxysuccinimidobiotin (NHS-biotin). Upon exposure of PSI complexes to a low concentration of endoproteinase glutamic acid (Glu)-C, PsaE was cleaved to 7.1- and 6.6-kD N-terminal fragments without significant cleavage of other subunits. Glu63 and Glu67, located near the C terminus of PsaE, were the most likely cleavage sites. At higher protease concentrations, the PsaE fragments were further cleaved and an N-terminal 9.8-kD PsaD fragment accumulated, demonstrating the accessibility of Glu residue(s) in the C-terminal domain of PsaD to the protease. Besides these major, primary cleavage products, several secondary cleavage sites on PsaD, PsaE, and PsaF were also identified. PsaF resisted proteolysis when PsaD and PsaE were intact. Glu88 and Glu124 of PsaF became susceptible to endoproteinase Glu-C upon extensive cleavage of PsaD and PsaE. Modification of PSI proteins with NHS-biotin and subsequent cleavage by endoproteinase Glu-C or thermolysin showed that the intact PsaE and PsaD, but not their major degradation products lacking C-terminal domains, were heavily biotinylated. Therefore, lysine-74 at the C terminus of PsaE was accessible for biotinylation. Similarly, lysine-107, or lysine-118, or both in PsaD could be modified by NHS-biotin.

Organization of photosystem I polypeptides examined by chemical cross-linking.

Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 was examined using the chemical cross-linkers glutaraldehyde and N-ethyl-1–3–[3-(dimethylamino)propyl]carbodiimide to investigate the organization of the polypeptide subunits. Thylakoid membranes and photosystem I, which was isolated by Triton X-100 fractionation, were treated with cross-linking reagents and were resolved using a Tricine/urea low-molecular-weight resolution gel system. Subunit-specific antibodies and western blotting analysis were used to identify the components of cross-linked species. These analyses identified glutaraldehyde-dependent cross-linking products composed of small amounts of PsaD and PsaC, PsaC and PsaE, and PsaE and PsaF. The novel cross-link between PsaE and PsaF was also observed following treatment with N-ethyl-1–3–[3-(dimethylamino)propyl]carbodiimide. These cross-linking results suggest a structural interaction between PsaE and PsaF and predict a trans-membrane topology for PsaF.

Evidence for two sites of inhibition of photosynthetic electron transport by dibromothymoquinone

Two sites in the photosynthetic electron transport chain of spinach chloroplasts are sensitive to inhibition by the plastoquinone antagonist dibromothymoquinone (2,5-dibromo3-methyl-&isopropyl-p-benzoquinone). This compound imposes maximal inhibition on reactions involving electron transport from water to a terminal acceptor such as ferricyanide at concentrations of about 1 PM. At concentrations of about 10 PM, dibromothymoquinone also inhibits electron transport reactions catalyzed by photosystem II in the presence of pphenylenediimines or p-benzoquinones. This inhibition is observed in both untreated and KCN/Hg-inhibited chloroplast preparations. Thiol incubation of chloroplasts exposed to dibromothymoquinone relieves inhibition at both sites. This reversal of inhibition is, however, different for the two sites. Restoration of ferricyanide reduction, which is blocked by 1 pM dibromothymoquinone, required high thiol/inhibitor ratios and incubation times with thiol of up to 3 min. The reversal of inhibition of p-phenylenediimine reduction by photosystem II, on the other hand, requires a thiol/inhibitor ratio of 1, and incubation times as short as 5 s. Addition of bovine serum albumin to absorb dibromothymoquinone results in a partial restoration of photosystem II reactions, but ferricyanide reduction, which requires photosystem II and photosystem I, cannot be restored by this procedure.

Reversal of dibromothymoquinone inhibition of photosynthetic electron transport by bovine serum albumin

Abstract Addition of bovine serum albumin to cholorplasts inhibited by prior addition of 1 μ m dibromothymoquinone results in a time- and light-dependent restoration of electron transport activity. The kinetics of this reversal reaction are complex, and indicate that it is controlled by the degree to which the thylakoid membranes are energized. The presence of ADP and inorganic phosphate, or of uncouplers, serves to retard the rate of reversal, whereas an acceleration of reversal is observed if the thylakoid membranes have been intentionally unstacked by exposure to low-salt medium. The reversal reaction reported here is unique to bovine serum albumin, and does not require the function of the free sulfhydryl group on the protein. It is concluded that the site of DBMIB inhibition associated with chloroplast membranes is situated in a position whose access to contact by bovine serum albumin is regulated by the structural changes induced by illumination and energization.

Steady-state kinetic analyses of Photosystem II activity catalyzed by lipophilic electron acceptors

Abstract Steady-state kinetic analyses of the Photosystem II activity elicited by quinones and quinonediimines in KCN/Hg-inhibited chloroplasts reveal that: 1. 1. Quinones generate a single reaction in which one quinone competes with another for photoreduction by Photosystem II. 2. 2. Quinonediimines also compete with one another for photoreduction, but the electron transport activity elicited by quinonediimines is the composite of two separate reactions, one of which is sensitive to ionic strength and atebrinbinding. 3. 3. The relationship between a quinone and a quinonediimine with respect to photoreduction by Photosystem II is non-competitive rather competitive. These findings are interpreted to indicate that quinones and quinonediimines accept electrons from two separate sites near the reducing site of Photosystem II.