Barbara A. Zilinskas

Salt and oxidative stress : similar and specific responses and their relation to salt tolerance in Citrus

Abstract. Salt damage to plants has been attributed to a combination of several factors including mainly osmotic stress and the accumulation of toxic ions. Recent findings in our laboratory showed that phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme active in the cellular antioxidant system, was induced by salt in citrus cells and mainly in roots of plants. Following this observation we studied the two most important enzymes active in elimination of reactive oxygen species, namely, superoxide dismutase (SOD) and ascorbate peroxidase (APX), to determine whether a general oxidative stress is induced by salt. While Cu/Zn-SOD activity and cytosolic APX protein level were similarly induced by salt and methyl viologen, the response of PHGPX and other APX isozymes was either specific to salt or methyl viologen, respectively. Unlike PHGPX, cytosolic APX and Cu/Zn-SOD were not induced by exogenously added abscisic acid. Salt induced a significant increase in SOD activity which was not matched by the subsequent enzyme APX. We suggest that the excess of H2O2 interacts with lipids to form hydroperoxides which in turn induce and are removed by PHGPX. Ascorbate peroxidase seems to be a key enzyme in determining salt tolerance in citrus as its constitutive activity in salt-sensitive callus is far below the activity observed in salt-tolerant callus, while the activities of other enzymes involved in the defence against oxidative stress, namely SOD, glutathione reductase and PHGPX, are essentially similar.

Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase

A cDNA clone encoding the cytosolic ascorbate peroxidase of pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. While ascorbate peroxidase shares limited overall homology with other peroxidases, significant homology with all known peroxidases was found in the vicinity of the putative active site.

Overexpression of Copper/Zinc Superoxide Dismutase in the Cytosol of Transgenic Tobacco Confers Partial Resistance to Ozone-Induced Foliar Necrosis.

Ozone damage to plants has been attributed to the action of oxygen free-radicals and other ozone degradation products against which cellular antioxidant systems have been considered to be a front-line defense. The activity of superoxide dismutase (SOD), one such antioxidant, has been shown to increase in ozonated plants. Past work with pea (Pisum sativum L.) in our laboratory showed that the cytosolic Cu/Zn-SOD isoform and its transcript were most responsive to ozone, compared to chloroplastic Cu/Zn-SOD. In the current work we tested the hypothesis that plants that constitutively overexpress cytosolic SOD are more tolerant of ozone. Pea cytosolic Cu/Zn-SOD was overproduced in the cytosol of two cultivars of transformed tobacco (Nicotiana tabacum), Bel W3 and Wisconsin 38. Young and recently expanded leaves of transgenic plants of both cultivars showed less foliar necrosis than nontransformed controls when exposed to acute doses of ozone. We suggest that this may demonstrate the importance of Cu/Zn-SOD in the cytosol as a protector of the integrity of the plasma membrane and possibly other cellular constituents.

Phycobilisome structure and function.

Phycobilisomes are aggregates of light-harvesting proteins attached to the stroma side of the thylakoid membranes of the cyanobacteria (blue-green algae) and red algae. The water-soluble phycobiliproteins, of which there are three major groups, tetrapyrrole chromophores covalently bound to apoprotein. Several additional protiens are found within the phycobilisome and serve to link the phycobiliproteins to each other in an ordered fashion and also to attach the phycobilisome to the thylakoid membrane. Excitation energy absorbed by phycoerythrin is transferred through phycocyanin to allophycocyanin with an efficiency approximating 100%. This pathway of excitation energy transfer, directly confirmed by time-resolved spectroscopic measurements, has been incorporated into models describing the ultrastructure of the phycobilisome. The model for the most typical type of phycobilisome describes an allophycocyanin-containing core composed of three cylinders arranged so that their longitudinal axes are parallel and their ends form a triangle. Attached to this core are six rod structures which contain phycocyanin proximal to the core and phycoerythrin distal to the core. The axes of these rods are perpendicular to the longitudinal axis of the core. This arrangement ensures a very efficient transfer of energy. The association of phycoerythrin and phycocyanin within the rods and the attachment of the rods to the core and the core to the thylakoid require the presence of several ‘linker’ polypeptides. It is recently possible to assemble functionally and structurally intact phycobilisomes in vitro from separated components as well as to reassociate phycobilisomes with stripped thylakoids. Understanding of the biosynthesis and in vivo assembly of phycobilisomes will be greatly aided by the current advances in molecular genetics, as exemplified by recent identification of several genes encoding phycobilisome components.Combined ultrastructural, biochemical and biophysical approaches to the study of cyanobacterial and red algal cells and isolated phycobilisome-thylakoid fractions are leading to a clearer understanding of the phycobilisome-thylakoid structural interactions, energy transfer to the reaction centers and regulation of excitation energy distribution. However, compared to our current knowledge concerning the structural and functional organization of the isolated phycobilisome, this research area is relatively unexplored.

Cloning, nucleotide sequence and mutational analysis of the gene encoding the photosystem II manganese-stabilizing polypeptide of Synechocystis 6803

SummaryAffinity purified, polyclonal antibodies raised against the Photosystem II 33 kDa manganese-stabilizing polypeptide of the spinach oxygen-evolving complex were used to isolate the gene encoding the homologous protein from Synechocystis 6803. Comparison of the amino acid sequence deduced from the Synechocystis psb1 nucleotide sequence with recently published sequences of spinach and pea confirms the homology indicated by antigenic crossreactivity and shows that the cyanobacterial and higher plant sequences are 43% identical and 63% conserved. Regions of identity, varying in length from 1 to 10 consecutive residues, are distributed throughout the protein. The 28 residues at the amino terminus of the psb1 gene product, characteristic of prokaryotic signal peptides, show homology with the carboxyl-terminal third of the transit sequences of pea and spinach and are most likely needed for the transport of the manganese-stabilizing protein across the thylakoid membrane to its destination of the lumen. Synechocystis mutants which contain a kanamycin resistance gene cassette inserted into the coding region for the 32 kDa polypeptide were constructed. These mutants contain no detectable 32 kDa polypeptide, do not evolve oxygen, and are incapable of photoautotrophic growth.

Kinetic and spectral properties of pea cytosolic ascorbate peroxidase

Sufficient highly purified native pea cytosolic ascorbate peroxidase was obtained to characterize some of its kinetic and spectral properties. Its rate constant for compound I formation from reaction with H2O2 is 4.0 × 107 M−1 s−1, somewhat faster than is typical for peroxidases. Compound I has the typical optical spectrum of an iron(IV)‐porphyrin‐π‐cation radical, despite considerable homology with yeast cytochrome c peroxidase. The rate constant for compound I reduction by ascorbate is extremely fast (8.0 × 107 M−1 s−1 at pH 7.8), again in marked contrast to the behavior of the yeast enzyme. The pH‐rate profile for compound I formation indicates a pK a value of 5.0 for a group affecting the active site reaction.

Spectral analysis of allophycocyanin I, II, III and B from Nostoc sp. phycobilisomes

Low temperature (-196C) and room temperature (25C) absorption spectra of a family of allophycocyanin spectral forms isolated from Nostoc sp. phycobilisomes as well as of the phycobilisomes themselves have been analyzed by Gaussian curve-fitting. Allophycocyanin I and B share long wavelength components at 668 and 679 nm, bands that are absent from allophycocyanin II and III. These long wavelength absorption components are apparently responsible for the 20 nm difference between the 680 nm fluorescence emission maximum of allophycocyanin I and B and the 660 nm maximum of II and III. This indicates that allophycocyanin I and B are the final acceptors of excitation energy in the phycobilisome and the excitation energy transfer bridge linking the phycobilisome with the chlorophyll-containing thylakoid membranes. These Gaussian components are also found in resolved spectra of phycobilisomes, are arguing against this family of allophycocyanin molecules being artifactual products of protein purification procedures.

Activated Oxygen Species in Multiple Stress Situations and Protective Systems

The electron configuration of molecular oxygen is unusual. It has two unpaired electrons, each in a π* orbital and having parallel spins, and thus it is a triplet molecule in the ground state. This is in contrast to most other molecules in the cell, which exist in the singlet ground state where all electrons have paired spins. Reactions between molecular oxygen and most molecules are therefore forbidden because of spin restriction. Molecular oxygen, however, can be converted to activated oxygen species by overcoming the spin restriction by a spin flip producing singlet oxygen (O2 1), or by the addition of either one, two or three electrons to form, respectively, the superoxide radical (O2 -), hydrogen peroxide (H2O2) or the hydroxyl radical (OH). Unlike molecular oxygen, these activated oxygen species (AOS) can be very reactive and are often referred to as reactive oxygen species (ROS). Cells must have effective mechanisms to remove excess AOS, particularly the most highly reactive hydroxyl radicals, to prevent oxidative damage to cellular components.

Comparative Immunology of the Phycobilisome Linker Polypeptides

Since the discovery of linker polypeptides by Tandeau de Marsac and Cohen-Bazire (1), these proteins have been found in all cyanobacterial and red algal phycobi 1 isome (PBsomes) examined. They are critical to the ordered assembly of this very efficient light harvesting antenna. They can be grouped into those polypeptides essential for the assembly of the PBsome rods and rod to core attachment and those needed for the association of allophycocyanin (APC) into the core and its attachment to the thylakoid (2,3). We have identified the function of these linker polypeptides in Nostoc sp. These PBsomes, not atypical of other cyanobacteria, are composed of an APC core, phycoerythrin- (PE) and phycocyanin- (PC) containing rods and five additional polypeptides of 95, 34.5, 34, 32 and 29 kD. The 95 kD polypeptide anchors the PBsome to the thylakoid membrane and is the direct mediator of excitation energy transfer from the PBsome to chl a (4); the 29 kD polypeptide attaches the rods to the core (5). in cells grown in cool white fluorescent light, the 32 kD polypeptide links two PE hexamers, and the 34 kD polypeptide mediates association of PE and with PC hexamers. in red light adapted cells, levels of PE and the associated 32 and 34 kD polypeptides are much reduced; in their stead are rods composed of PC and a 34.5 kD polypeptide which serves to link PC hexamers (6).

Orientation and linear dichroism of Mastigocladus laminosus phycocyanin trimer and Nostoc sp. phycocyanin dodecamer in stretched poly(vinyl alcohol) films

The linear dichroism (LD) spectra of the C-phycocyanin (C-PC) trimer disks oriented in poly(vinyl alcohol) films (PVA) at room temperature and at 95 K were determined. Utilizing the known atomic coordinates of the chromophores (Schirmer, T., Bode, W. and Huber, R. (1987) J. Mol. Biol. 196, 677-695) and theoretical estimates of the orientations of the transition dipole moments relative to the molecular framework, the LD spectra were simulated using the pairwise exciton interaction model of Sauer and Scheer (Biochim. Biophys. Acta 936 (1988) 157-170); in this model, the alpha 84 and beta 84 transition moments are coupled by an exciton mechanism, while the beta 155 chromophore remains uncoupled. Linear dichroism spectra calculated using this exciton model, as well as an uncoupled chromophore (molecular) model, were compared with experimental LD spectra. Satisfactory qualitative agreement can be obtained in both the exciton and molecular models using somewhat different relative values of the theoretically estimated magnitudes of the beta 155 oscillator strength. Because the relative contributions of each of the chromophores (and thus exciton components) to the overall absorption of the C-PC trimer are not known exactly, it is difficult to differentiate successfully between the molecular and exciton models at this time. The linear dichroism spectra of PC dodecamers derived from phycobilisomes of Nostoc sp. oriented in stretched PVA films closely resemble those of the C-PC trimers from Mastigocladus laminosus, suggesting that the phycocyanin chromophores are oriented in a similar manner in both cases, and that neither linker polypeptides nor the state of aggregation have a significant influence on these orientations and linear dichroism spectra. The LD spectra of oriented phycocyanins in stretched PVA films at low temperatures (95 K) appear to be of similar quality and magnitude as the LD spectra of single C-PC crystals (Schirmer, T. and Vincent, M.G. (1987) Biochim. Biophys. Acta 893, 379-385).