Subunit composition of Photosystem I complex that catalyzes light-dependent transfer of electrons from plastocyanin to ferredoxin.

The PSI core complex prepared from cucumber cotyledons, which contains 80 chlorophylls per reaction center (P700) and eight polypeptides with apparent molecular masses of 65/63, 20, 19.5, 18.5, 17.5, 7.6, and 5.8 kDa, has been shown to catalyze the light-dependent transfer of electrons from plastocyanin to ferredoxin. The "native" PSI complex, which contains more than fifteen polypeptides and 120 chlorophylls per P700, did not show higher activity. Any attempt to deplete subunit(s) of the core complex decreased its activity. These results suggest that in addition to light-harvesting chlorophyll a/b protein complexes, several genes of psaA-psaK, which have been proposed as components of PSI complex, are not involved in the activity of PSI complex. It was also found that the amount of 18.5-kDa polypeptide in the PSI complex affects the activity: when this polypeptide was largely depleted, the complex was almost inactive. The inactivation was due to inhibition of electron transfer from plastocyanin to photooxidized P700. Chemical cross-linking and N-terminal amino acid sequencing experiments indicated that the 18.5-kDa polypeptide is the plastocyanin-docking protein and the psaF gene product. The function of the psaF gene product was discussed.     

Interaction of plastocyanin with oligopeptides: effect of lysine distribution within the peptide

We synthesized and purified four oligopeptides containing four lysines (KKKK, GKKGGKK, KKGGGKK, and KGKGKGK) as models for the plastocyanin (PC) interacting site of cytochrome f. These peptides competitively inhibited electron transfer between cytochrome c and PC. The inhibitory effect increased as the peptide concentrations were increased. The association constants between PC and the peptides did not differ significantly (3500-5100 M(-1)), although the association constant of PC-KGKGKGK was a little larger than the constants between PC and other peptides. Changes in the absorption spectrum of PC were observed when the peptides were added to the PC solution: peaks and troughs were detected at about 460 and 630 nm and at about 560 and 700 nm, respectively, in the difference absorption spectra between the spectra with and without peptides. These changes were attributed to the structural change at the copper site of PC by interaction with the peptides. The structural change was most significant when tetralysine was used. These results show that binding of the oligopeptide to PC is slightly more efficient when lysines are distributed uniformly within the peptide, whereas the structural change of PC becomes larger when the lysines are close to each other within the peptide.     

Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and Arabidopsis plants

Transgenic tobacco plants expressing the ascorbate oxidase (AAO) gene in sense and antisense orientations, and an Arabidopsis mutant in which the T-DNA was inserted into a putative AAO gene, were used to examine the potential roles of AAO for salt-stress tolerance in plants. AAO activities in the transgenic tobacco plants expressing the gene in sense and antisense orientations were, respectively, about 16-fold and 0.2-fold of those in the wild type. Under normal growth conditions, no significant differences in phenotypes were observed, except for a delay in flowering time in the antisense plants. However, at high salinity, the percentage germination, photosynthetic activity, and seed yields were higher in antisense plants, with progressively lower levels in the wild type and the sense plants. The redox state of apoplastic ascorbate in sense plants was very low even under normal growth conditions. Upon salt stress, the redox state of symplastic and apoplastic ascorbate decreased among the three types of plants, but was lowest in the sense plants. The hydrogen peroxide contents in the symplastic and apoplastic spaces were higher in sense plants, progressively lower in the wild type, followed by the antisense plants. The Arabidopsis T-DNA inserted mutant exhibited very low ascorbate oxidase activity, and its phenotype was similar to that of antisense tobacco plants. These results suggest that the suppressed expression of apoplastic AAO under salt-stress conditions leads to a relatively low level of hydrogen peroxide accumulation and a high redox state of symplastic and apoplastic ascorbate which, in turn, permits a higher seed yield.     

Functional Differences Between Cyanobacterial DnaK1 Chaperones from the Halophyte Aphanothece halophytica and the Freshwater Species Synechococcus elongatus Expressed in Escherichia coli

DnaK chaperones participate in essential cellular processes including the assistance of the folding, structural maintenance, trafficking, and degradation of proteins, the control of stress responses, and so on. In contrast to the situation found in most other bacterial groups, the cyanobacteria contain multiple dnaK homolog genes whose cellular roles remain ambiguous. We compared in this work the in vivo chaperone capabilities of the DnaK1 members from the halophyte Aphanothece halophytica and the freshwater species Synechococcus elongatus. The corresponding dnaK1 genes were expressed in Escherichia coli, and the abilities of the encoded chaperones to provide for both general and specific functions conducted by E. coli DnaK were analyzed. Synechococcus DnaK1 was far more effective than A. halophytica DnaK1 in replacing E. coli DnaK in all activities tested in vivo, including changes in cell morphology and downregulation of the heat shock response, prevention of the aggregation of misfolded proteins, and restoration of thermotolerance to dnaK-deficient mutants. Thus, regardless of an extensive sequence similarity and comparable in vitro chaperone capabilities, the two cyanobacterial DnaK1 chaperones functionally differed under in vivo conditions. The overall results reinforce the notion that A. halophytica DnaK1 and Synechococcus DnaK1 evolved different substrate specificity since they separated from a common ancestor.     

Electron Flow from NAD(P)H Dehydrogenase to Photosystem I is Required for Adaptation to Salt Shock in the Cyanobacterium Synechocystis sp. PCC 6803

The role of the NAD(P)H-dehydrogenase complex in adaptationto salt stress was examined in an ndhB-inacti-vated mutant ofthe cyanobacterium Synechocystis sp. PCC 6803. Wild-type cellsand ndhB-inactivated mutant cells grew at similar rates underconditions of low salinity (<0.6M NaCl) and high CO² (3%).However, when the concentration of NaCl in the culture mediumwas higher than 0.6 M, the mutant cells grew much more slowlythan the wild-type cells. Upon addition of high concentrationsof NaCl, the oxygen-evolving activity was rapidly inhibitedbut then it recovered, with the rate of recovery depending onthe concentration of NaCl. The recovery of the mutant cellswas significantly delayed when the concentration of NaCl wasabove 0.3 M. At 0.9 M NaCl, wild-type cells recovered with ahalf time of about 40 min, while mutant cells did not recover.The kinetics of changes in Chi fluorescence confirmed theseresults. In wild-type cells, input of electrons from the cytosolto PSI via the NAD(P)H-dehy-drogenase complex increased uponsalt shock. It appears, therefore, that the electron flow fromthe cytosol to PSI via NAD(P)H-dehydrogenase is essential forthe adaptation of cyanobacteria to salt shock. (Received June 11, 1997; Accepted September 24, 1997)     

Direct Visualization of Lignifying Secondary Wall Thickenings in Zinnia elegansCells in Culture

The lignifying secondary wall thickenings of tracheary elementsthat were differentiating from Zinnia mesophyll cells in suspensionculture were examined by a freeze-etch replica technique. Cellulosemicrofibrils in primary and secondary wall thickenings differedin terms of both width and arrangement. The primary wall wasobserved as a randomly arranged network of microfibrils. Bycontrast to microfibrils in the secondary wall thickenings werehighly organized, with many pores and spaces between them. Numerousfilamentous and granular cross-links were observed in both primarywalls and secondary wall thickenings. As lignifica-tion proceeded,the cellulose microfibrils in secondary wall thickenings becameobscure as a result of the deposition of large numbers of sphericalbodies around and between the microfibrils. This material hadcompletely covered the fibrous matrix by the end of lignification.It might have been composed of the products of the dehydrogenationof monolignols. We also noted that the microfibrils appearedto be slightly irregular or wavy just after the start of lignificationbut were straighter and appeared to be more rigid when lignificationwas complete. (Received July 25, 1996; Accepted April 24, 1997)