Preferential induction of a 9-lipoxygenase by salt in salt-tolerant cells of Citrus sinensis L. Osbeck.

Recent findings in our laboratory suggested that in citrus cells the salt induction of phospholipid hydroperoxide glutathione peroxidase, an enzyme active in cellular antioxidant defense, is mediated by the accumulation of hydroperoxides. Production of hydroperoxides occurs as a result of non-enzymatic auto-oxidation or via the action of lipoxygenases (LOXs). In an attempt to resolve the role of LOX activity in the accumulation of peroxides we analyzed the expression of this protein under stress conditions and in cells of Citrus sinensis L. differing in sensitivity to salt. Lipoxygenase expression was induced very rapidly only in the salt-tolerant cells and in a transient manner. The induction was specific to salt stress and did not occur with other osmotic-stress-inducing agents, such as polyethylene glycol or mannitol, or under hot or cold conditions, or in the presence of abscisic acid. The induction was eliminated by the antioxidants dithiothreitol and kaempferol, thus once more establishing a correlation between salt and oxidative stresses. Analyses of both in vitro and in vivo products of LOX revealed a specific 9-LOX activity, and a very fast reduction of the hydroperoxides to the corresponding hydroxy derivatives. This suggests that one of the metabolites further downstream in the reductase pathway may play a key role in triggering defense responses against salt stress.

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

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.

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

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.

Induction of a gene encoding an oleosin homologue in cultured citrus cells exposed to salt stress.

A cDNA clone (C3) with high homology to plant oleosins was isolated from citrus cultured cells. The 827-bp cDNA insert has an open reading frame of 144 amino-acid residues. The central hydrophobic domain of the protein is nearly identical to oleosins from Brassica napus and maize, and the C-terminal hydrophilic region following the hydrophobic domain is also highly conserved. The steady-state level of mRNA hybridizing to C3 was significantly increased upon exposure of citrus cells to 0.2 M NaCl. A lower level of transcript was found in seeds, but none could be detected in any other vegetative tissue (leaves, roots or fruit) even in the presence of salt under the conditions used. The induction of the oleosin homologue in citrus cells by salt does not depend on the developmental stage of the cells.

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.

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.

Changing root system architecture through inhibition of putrescine and feruloyl putrescine accumulation.

Plant roots provide anchorage and absorb the water and minerals necessary for photosynthesis in the aerial parts of the plant. Since plants are sessile organisms, their root systems must forage for resources in heterogeneous soils through differential branching and elongation [(1988) Funct. Ecol. 2, 345-351; (1991) Plant Roots: The Hidden Half, pp. 3-25, Marcel Dekker, NY]. Adaptation to drought, for instance, can be facilitated by increased root growth and penetration. Root systems thus develop as a function of environmental variables and the needs of the plant [(1988) Funct. Ecol. 2, 345-351; (1986) Bot. Gaz. 147, 137-147; (1991) Plant Roots: The Hidden Half, pp. 309-330, Marcel Dekker, NY]. We show, in a model system consisting of excised tobacco roots, that both alpha-DL-difluoromethylornithine (an inhibitor of putrescine biosynthesis) and the rolA gene (from the root-inducing transferred DNA of Agrobacterium rhizogenes) stimulate overall root growth and cause a conversion in the pattern of root system formation, producing a dominant or 'tap' root. These morphological changes are correlated with a depression in the accumulation of polyamines and their conjugates.

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 A2 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.

A novel plant glutathione peroxidase-like protein provides tolerance to oxygen radicals generated by paraquat in Escherichia coli.

Citrus salt-stress associated protein (Cit-SAP) reveals significant sequence homology to mammalian glutathione peroxidase (GP). In an attempt to assign biological function to this protein, transformed E. coli cells expressing Cit-SAP were examined for their ability to tolerate free radicals formed by paraquat, an O2- radical forming agent. In the presence of paraquat, the survival rate of the transformed bacteria expressing Cit-SAP was much higher as compared to the wild-type bacteria. The results support the assumption that Cit-SAP is a plant GP-like protein which participate in the enzymatic system aimed at scavenging oxygen free-radicals in plants.

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.

Plantlet regeneration from a NaCl-selected salt-tolerant callus culture of Shamouti orange (Citrus sinensis L. Osbeck).

Plantlets were regenerated from a selected salt-tolerant cell line of Shamouti orange (Citrus sinensis L. Osbeck). Embryogenesis was carried out both in the presence and absence of NaCl, yielding green and white globular embryos, respectively. Greening could be induced subsequently and normal heart shape embryo development was obtained. Plantlet formation required exposure to kinetin prior to the introduction of the root-inducing hormone naphthalene acetic acid. This system differs from the designed protocol for plant regeneration from the salt-sensitive, i.e., unselected callus. It is concluded that NaCl interferes with the regeneration process, with embryogenesis and/or embryo development into plantlets. Its presence during callus growth probably changes the balance of the phytohormones which is later manifested in plant regeneration. Citrus salt-tolerant callus yields salt-tolerant embryos. Salt-tolerant calli derived from regenerated plantlets indicate acquisition of salt tolerance on the whole plant level.

Relationship between Salt Tolerance and Resistance to Polyethylene Glycol-Induced Water Stress in Cultured Citrus Cells.

Salt-tolerant selected cells of Shamouti orange (Citrus sinensis) and Sour orange (Citrus aurantium) grew considerably better than nonselected cells at any NaCl concentration tested up to 200 millimolar. Also, the growth response of each treatment was identical in the two species. However, the performance of cells of the two species under osmotic stress induced by polyethylene glycol (PEG), which is presumably a nonabsorbed osmoticum, was significantly different. The nonselected Shamouti cell lines were significantly more sensitive to osmotic stress than the selected cells. The salt adapted Shamouti cells were apparently also adapted to osmotic stress induced by PEG. In Sour orange, however, the selected lines had no advantage over the nonselected line in response to osmotic stress induced by PEG. This response was also similar quantitatively to the response of the selected salt-tolerant Shamouti cell line. It seems that the tolerance to salt in Shamouti, a partial salt excluder, involves an osmotic adaptation, whereas in Sour orange, a salt accumulator, such an adaptation apparently does not occur. PEG-induced osmotic stress causes an increase in the percent dry weight of salt-sensitive and salt-tolerant cells of both species. No such increase was found under salt stress. The size of control and stressed cells is not significantly different.

Role of Internal Potassium in Maintaining Growth of Cultured Citrus Cells on Increasing NaCl and CaCl(2) Concentrations.

Shamouti orange (Citrus sinensis L. Osbeck) salt-tolerant cells were grown under low water potential conditions induced by polyethylene glycol (PEG), NaCl, and CaCl(2). On the basis of equal osmotic potentials, PEG was the least inhibitory, NaCl next, and CaCl(2) the most inhibitory. The relation between growth capacity and ion content can be summarized as follows. (a) Internal K(+) concentration was a major factor which changed in the presence of PEG, NaCl, and CaCl(2) and probably played a key role in determining growth capacity. (b) Internal concentrations of Na(+), Ca(2+), or Cl(-) could not be directly correlated with growth. (C) Internal Mg(2+) concentration could be significant only in the presence of high external Ca(2+) concentrations. (d) The contribution of nitrate and phosphate to the internal osmoticum was negligible. The ratio of external (Ca(2+))/(Na(+))(2) concentration is crucial for growth. Ratios above 0.5 x 10(-4) per millimolar gave maximal protection from adverse effects of NaCl. Growth capacity was found to be determined by the combination of (Ca(2+))/(Na(+))(2) ratio and the absolute external concentration of NaCl. However, a correlation between internal K(+) concentration and growth capacity seemed independent of external NaCl concentration.

Relation between Ion Accumulation of Salt-Sensitive and Isolated Stable Salt-Tolerant Cell Lines of Citrus aurantium.

Four selected NaCl-tolerant cell lines of Sour orange (Citrus aurantium) were compared with the nonselected cell line in their growth and internal ion content of Na(+), K(+), and Cl(-) when exposed to increasing NaCl concentrations. No difference was found among the various NaCl-tolerant cell lines in Na(+) and Cl(-) uptake, and all these cell lines took up similar or even larger amounts of Na(+) and Cl(-) than the NaCl-sensitive cell line. Exposure of cells of NaCl-sensitive and NaCl-tolerant lines to equal external concentrations of NaCl, resulted in a greater loss of K(+) from the NaCl-sensitive cell line. This observation leads to the conclusion that growth and ability to retain high levels of internal K(+) are correlated. Exposure of the NaCl-tolerant cell lines to salts other than NaCl resulted in even greater tolerance to Na(2)SO(4), but rather poor tolerance to K(+) introduced as either K(2)SO(4) or KCl; the latter has a stronger inhibitory effect. The NaCl-sensitive cell line proved to be more sensitive to replacement of Na(+) by K(+). Analyses of internal Na(+), K(+), and Cl(-) concentrations failed to identify any particular internal ion concentration which could serve as a reliable marker for salt tolerance.

Aspects of Salt Tolerance in a NaCl-Selected Stable Cell Line of Citrus sinensis.

A NaCl-tolerant cell line which was selected from ovular callus of ;Shamouti' orange (Citrus sinensis L. Osbeck) proved to be a true cell line variant. This conclusion is based on the following observations. (a) Cells which have been removed from the selection pressure for at least four passages retain the same NaCl tolerance as do cells which are kept constantly on 0.2 molar NaCl. (b) Na(+) and Cl(-) uptake are considerably lower in salt-tolerant cells (R-10) than in salt-sensitive cells (L-5) at a given external NaCl concentration. (c) Growth of salt-tolerant cells is markedly suppressed upon replacement of NaCl by KCl, whereas the growth of salt-sensitive cells is only slightly affected. Accumulation of K(+) and Cl(-) accompanies the inhibition of growth. Experiments carried out with sodium and potassium sulfate suggest that the toxic effect is due to the accumulated Cl(-). (d) Removal of Ca(2+) from the growth medium severely inhibits the growth of salt-tolerant cells in the presence of NaCl, while it has a minor effect on growth of salt-sensitive cells in the presence of NaCl. (e) Electron micrographs show that the salt-tolerant cells have very big vacuoles when exposed to salt, while the size of the vacuoles of the salt-sensitive cells does not change.

Evaluation of isozyme systems in Citrus to facilitate identification of fusion products.

Ten isozymes were analyzed in nucellar calli of nine Citrus species and cultivars and roots of the corresponding apomictic seedlings. The zymograms obtained can be divided into three groups: a) isozyme patterns similar in both calli and roots, b) isozyme patterns similar in calli but variable in roots, and c) isozyme patterns variable in both calli and roots. Analysis of these ten isozyme systems may facilitate identification of fusion products in Citrus.

Mg2+ translocation across the thylakoid membrane: studies using the ionophore A 23187.

1. The degree of the light-dependent Mg2+ efflux across the thylakoid membrane is a function of pH. There is a considerable efflux at pH 7.2 which decreases to a negligible amount at pH 8.6. This conclusion is derived from studies using the divalent cation-specific ionophore A23187. The ionophore is active as an uncoupler at pH 7.2 even in the absence of added Mg2+, and completely inactive at pH 8.6 unless Mg2+ is added. The activity was assayed as the effect on both the rate of electron transport (stimulation at pH 7.2 and inhibition at pH 8.6) and deltapH. 2. Under conditions of maximal Mg2+ efflux, pH 7.2, and in the presence of EDTA, it is shown that Mg2+ is transported across the thylakoid membrane, but it is not diluted in the medium. 3. At high pH, light-induced proton influx is not compensated by Mg2+ efflux in the presence of a relatively permeable anion. However, in the presence of an impermeable anion, where cation efflux is necessary, Mg2+ efflux occurs, indicating its preference to K+ efflux. It is suggested that although light-induced Mg2+ efflux across the thylakoid membrane is evident, its magnitude is small and it is not diluted in the stroma. Thus, it seems hard to visualize how this transport is supposed to play an important role in CO2 fixation.

Proton translocation and ATP formation coupled to electron transport from H2O to the primary acceptor of photosystem 2.

1. The rate of electron transport from H2O to silicomolybdate in the presence of 3-(3-4-dichlorophenyl)-1,1-dimethylurea (diuron) (which involves the oxygen-evolving enzyme, the photochemistry of photosystem 2 and the primary electron acceptor of photosystem 2) is controlled by internal pH. This is based on the shift of the pH profile of the rate of electron transport upon addition of uncouplers, or by using EDTA-treated chloroplasts. Both stimulation and inhibition of electron transport by addition of uncouplers (depending on external pH) could be observed. These effects are obtained in the diuron-insensitive photoreductions of either silicomolybdate or ferricyanide. These experiments provide strong evidence that a proton translocating site exists in the sequence of the electron transport H2O leads to Q (the primary acceptor of photosystem 2). 2. The photoreduction of silicomolybdate in the presence of diuron causes the formation of delta pH. The value of delta pH depends on the external pH and its maximal value was shown to be 2.4. The calculated internal pH at different external pH values was found to be rather constant, namely between 5.1 -- 5.2. 3. Electron transport from H2O to silicomolybdate (in the presence of diuron) does not support ATP formation. It is suggested that this is due to the fact that the delta pH formed is below the "threshold" delta pH required for the synthesis of ATP. By adding an additional source of energy in the form of a dark diffusion potential created in the presence of K+ and valinomycin, significant amounts of ATP are formed in this system.