Gozal Ben-Hayyim

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 characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.

A stress‐associated citrus protein is a distinct plant phospholipid hydroperoxide glutathione peroxidase

A protein whose level is markedly increased upon exposure of cultured citrus cells and whole plants to NaCl, was shown to specifically catalyze the reduction of phosphatidylcholine hydroperoxide in the presence of glutathione. This enzymatic activity was shown to be independent of a similar activity exhibited by glutathione S‐transferase in plants. This finding corroborates the significant homology (52%) accounted between the deduced amino acid sequence of the gene encoding for this protein and that of mammalian phospholipid hydroperoxide glutathione peroxidases. While the mammalian enzyme is known and well investigated, this study establishes the presence of this key protein also in plants.

The Salt-Stress Signal Transduction Pathway That Activates the gpx1 Promoter Is Mediated by Intracellular H2O2, Different from the Pathway Induced by Extracellular H2O2

Several genes encoding putative glutathione peroxidase have been isolated from a variety of plants, all of which show the highest homology to the phospholipid hydroperoxide isoform. Several observations suggest that the proteins are involved in biotic and abiotic stress responses. Previous studies on the regulation of gpx1, the Citrus sinensis gene encoding phospholipid hydroperoxide isoform, led to the conclusion that salt-induced expression of gpx1 transcript and its encoded protein is mediated by oxidative stress. In this paper, we describe the induction of gpx1 promoter:uidA fusions in stable transformants of tobacco (Nicotiana tabacum) cultured cells and plants. We show that the induction of gpx1 by salt and oxidative stress occurs at the transcriptional level. gpx1 promoter analysis confirmed our previous assumption that the salt signal is transduced via oxidative stress. We used induction of the fusion construct to achieve better insight into, and to monitor salt-induced oxidative stress. The gpx1 promoter responded preferentially to oxidative stress in the form of hydrogen peroxide, rather than to superoxide-generating agents. Antioxidants abolished the salt-induced expression of gpx1 promoter, but were unable to eliminate the induction by H2O2. The commonly employed NADPH-oxidase inhibitor diphenyleneiodonium chloride and catalase inhibited the H2O2-induced expression of gpx1 promoter, but did not affect its induction by salt. Our results led us to conclude that salt induces oxidative stress in the form of H2O2, its production occurs in the intracellular space, and its signal transduction pathway activating the gpx1 promoter is different from the pathway induced by extracellular H2O2.

Selection of Stable Salt-Tolerant Callus Cell Lines and Embryos in Citrus sinensis and C. aurantium*

Summary Several callus cell lines of «Shamouti» (Citrus sinensis Osbeck) and one cell line of «Sour orange» (C. aurantium) capable of growing in the presence of up to about 10 g l-1 NaCl, were obtained by repeated exposure of the cultured ovular cells to a medium containing salt. Gamma irradiation prior to exposure to salt gave no selective advantage. Increased tolerance for salt was retained even after three consecutive transfers in medium without salt, indicating isolation of genetic variants exhibiting stability of the salt-tolerance trait. Replacing NaCl by KCl resulted in significantly increased sensitivity to salt. However, when Cl- was replaced by SO4-2, the NaCl-tolerant cell lines grew well on either Na2SO4 or K2SO4. The salt-sensitive line can be induced to undergo embryogenesis in the presence of galactose. The salt-tolerant cell lines still preserve the capacity to regenerate embryos, though to a different degree. Some of these lines regenerate embryos in the presence of galactose, while others require the combination of salt and galactose in the medium. Embryos regenerated from salt-tolerant callus cells survived and grew better in saline medium than did embryos regenerated from the original salt sensitive callus.

Regulation of stress-induced phospholipid hydroperoxide glutathione peroxidase expression in citrus

Abstract. Recent findings in our laboratory showed that in citrus cells, salt treatment induced the accumulation of mRNA and a protein corresponding to phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme active in the cellular antioxidant system. The protein and its encoding gene, csa, were isolated and characterized, and the expected enzymatic activity was demonstrated (G. Ben-Hayyim et al., 1993, Plant Sci. 88: 129–140; D. Holland et al., 1993, Plant Mol. Biol. 21: 923–927; D. Holland et al., 1994, FEBS Lett. 337: 52–55; T. Beeor-Tzahar et al., 1995, FEBS Lett. 366: 151–155). In an attempt to find out how salt induces the expression of an antioxidant enzyme, the regulation of PHGPX in citrus cells was studied at both the mRNA transcript and the protein levels. A high and transient response at the csa mRNA level was observed after 4–7 h of exposing salt-sensitive cells to NaCl, or abscisic acid, whereas no response could be detected in the salt-tolerant cells under the same conditions. tert-Butylhydroperoxide, a substrate of PHGPX, induced csa mRNA transcripts after only 2 h, and abolished the differential response between salt-sensitive and salt-tolerant cells. On the basis of these results and those obtained under heat and cold stresses, it is suggested that csa is directly induced by the substrate of its encoded enzyme PHGPX, and that salt induction occurs mainly via the production of reactive oxygen species and hydroperoxides.

Stimulatory Effect of Glycerol on Growth and Somatic Embryogenesis in Citrus Callus Cultures

Summary The effect of glycerol on growth and embryogenesis of nucellar calli from several Citrus cultivars was studied. Glycerol induces a high rate of growth in «Shamouti» and Sour orange, in «Duncan» grapefruit and «Villafranca» lemon. «Volckameriana» lemon and several mandarin cultivars either utilize glycerol rather poorly or not at all. Growth curves of calli capable of utilizing glycerol are characterized by a longer lag-period and somewhat slower growth rate as compared with the growth curves obtained with calli in a sucrose-containing medium. Growth of Citrus calli on glycerol is accompanied by somatic embryogenesis. Within three to four weeks of subculturing the callus in the presence of 280 mM glycerol, the whole culture is transformed into small green embryos.

Drought, heat and salt stress induce the expression of a citrus homologue of an atypical late-embryogenesis Lea5 gene.

In a search for genes that are induced in citrus cell suspension in response to salt stress, a cDNA clone with high homology to cotton Lea5 gene was isolated. Data base analysis of the protein deduced from the nucleotide sequence indicates that, like in cotton, the protein from citrus contains regions with significant hydropathic character. The gene, designated C-Lea5, is expressed in citrus leaves as well as cell suspension. The steady-state level of C-Lea5 is increased in cell suspension that is grown in the presence of 0.2 M NaCl. This phenomenon is also observed in leaves of citrus plants irrigated with NaCl and in citrus seedlings which are exposed to drought and heat stress. We suggest that the osmotic stress resulted from elevated level of salt is responsible for the increase in the level of C-Lea5.

Quantitative competition of calcium with sodium or magnesium for sorption sites on plasma membrane vesicles of melon (Cucumis melo L.) root cells

The presence of Ca2+ ions in solution is vital for root growth. The plasma membrane is one of the first sites where competition between Ca2+ and other ions occurs. We studied the competition between Ca2+ and Na+ or Mg2+ for sorption sites on the plasma membrane of melon root cells.Sorption of 45Ca2+ to right-side-out PM vesicles of melon (Cucumis melo L.) roots (prepared by aqueous two-phase partitioning) was studied at various Ca2+ concentrations, in the presence of increasing concentrations of Na+ or Mg2+ chlorides. Experimentally determined amounts of Ca2+ sorbed to the plasma membrane vesicles agreed fairly well with those calculated from a competitive sorption model. The best fit of the model to the experimental data was obtained for an average surface area of 370 Å2 per charge, and binding coefficients for Na+, Mg2+ and Ca2+ of 0.8, 9 and 50 m-1, respectively.Our results suggest that nonphospholipid components in the plasma membrane contribute significantly to Ca2+ binding. The high affinity of Ca2+ binding to the plasma membrane found in this study might explain the specific role of Ca2+ in relieving salt stress in plant roots.

Isolation and characterization of salt-associated protein in Citrus

Abstract A significant increase in the amount of a protein, whose migration in two-dimensional gel electrophoresis corresponds to an apparent olecular weight of 23–25 kDa and pI=6.1, was observed in adapted salt-tolerant cultured cells derived from Shamuti orange (Citrus sinesis L. Osbeck) ovular callus cells. An increase was also determined when these cells were grown in the presence of abscisic acid (ABA) or polyethylene glycol (PEG) instead of NaCl, similarly to what was previously observed for osmotin, the tobacco salt-associated protein. However, no similarity has been so far observed between these proteins in their biochemical and immunochemical properties. Pulse labeling of the salt-tolerant cells suggests a low turnover of the accumulated protein. Its salt-induced accumulation in the cells was also relatively slow and gradually increased during a period of several days following exposure of the cells to NaCl. Cell fractionation experiments suggest that the protein is allocated in the soluble fraction and is not an integral membrane protein. Examination of the presence of the protein in citrus plants, such as Etrog citron (C. medicaL.) and Cleopatra mandarin (C. reticulata) cultivars, revealed an increase in its concentration in all organs examined, following irrigation of the plants with saline water. Thus, this protein is associated with stress conditions employed on growing plants as well and is not a unique phenomenon restricted to cultured cells only.