Signal transduction networks and the biology of plant cells.

The development of plant transformation in the mid-1980s and of many new tools for cell biology, molecular genetics, and biochemistry has resulted in enormous progress in plant biology in the past decade. With the completion of the genome sequence of Arabidopsis thaliana just around the corner, we can expect even faster progress in the next decade. The interface between cell biology and signal transduction is emerging as a new and important field of research. In the past we thought of cell biology strictly in terms of organelles and their biogenesis and function, and researchers focused on questions such as, how do proteins enter chloroplasts? or, what is the structure of the macromolecules of the cell wall and how are these molecules secreted? Signal transduction dealt primarily with the perception of light (photomorphogenesis) or hormones and with the effect such signals have on enhancing the activity of specific genes. Now we see that the fields of cell biology and signal transduction are merging because signals pass between organelles and a single signal transduction pathway usually involves multiple organelles or cellular structures. Here are some examples to illustrate this new paradigm. How does abscisic acid (ABA) regulate stomatal closure? This pathway involves not only ABA receptors whose location is not yet known, but cation and anion channels in the plasma membrane, changes in the cytoskeleton, movement of water through water channels in the tonoplast and the plasma membrane, proteins with a farnesyl tail that can be located either in the cytosol or attached to a membrane, and probably unidentified ion channels in the tonoplast. In addition there are highly localized calcium oscillations in the cytoplasm resulting from the release of calcium stored in various compartments. The activities of all these cellular structures need to be coordinated during ABA-induced stomatal closure. For another example of the interplay between the proteins of signal transduction pathways and cytoplasmic structures, consider how plants mount defense responses against pathogens. Elicitors produced by pathogens bind to receptors on the plant plasma membrane or in the cytosol and eventually activate a large number of genes. This results in the coordination of activities at the plasma membrane (production of reactive oxygen species), in the cytoskeleton, localized calcium oscillations, and the modulation of protein kinases and protein phosphatases whose locations remain to be determined. The movement of transcription factors into the nucleus to activate the defense genes requires their release from cytosolic anchors and passage through the nuclear pore complexes of the nuclear envelope. This review does not cover all the recent progress in plant signal transduction and cell biology; it is confined to the topics that were discussed at a recent (November 1998) workshop held in Santiago at which lecturers from Chile, the USA and the UK presented recent results from their laboratories.

Electric signaling and pin2 gene expression on different abiotic stimuli depend on a distinct threshold level of endogenous abscisic acid in several abscisic acid-deficient tomato mutants

Experiments were performed on three abscisic acid (ABA)-deficient tomato (Lycopersicon esculentum Mill.) mutants, notabilis, flacca, and sitiens, to investigate the role of ABA and jasmonic acid (JA) in the generation of electrical signals and Pin2 (proteinase inhibitor II) gene expression. We selected these mutants because they contain different levels of endogenous ABA. ABA levels in the mutant sitiens were reduced to 8% of the wild type, in notabilis they were reduced to 47%, and in flacca they were reduced to 21%. In wild-type and notabilis tomato plants the induction of Pin2 gene expression could be elicited by heat treatment, current application, or mechanical wounding. In flacca and sitiens only heat stimulation induced Pin2 gene expression. JA levels in flacca and sitiens plants also accumulated strongly upon heat stimulation but not upon mechanical wounding or current application. Characteristic electrical signals evolved in the wild type and in the notabilis and flacca mutants consisting of a fast action potential and a slow variation potential. However, in sitiens only heat evoked electrical signals; mechanical wounding and current application did not change the membrane potential. In addition, exogenous application of ABA to wild-type tomato plants induced transient changes in membrane potentials, indicating the involvement of ABA in the generation of electrical signals. Our data strongly suggest the presence of a minimum threshold value of ABA within the plant that is essential for the early events in electrical signaling and mediation of Pin2 gene expression upon wounding. In contrast, heat-induced Pin2 gene expression and membrane potential changes were not dependent on the ABA level but, rather, on the accumulation of JA.

Inhibition of wound-induced accumulation of allene oxide synthase transcripts in flax leaves by aspirin and salicylic acid

Allene oxide synthase (AOS) mediates the conversion of lipoxygenase-derived fatty acid hydroperoxides to unstable allene epoxides, which supply the precursors for the synthesis of the phytohormone jasmonic acid (JA). In this study the characterization of AOS gene expression in flax (Linum usitatissimum) is reported. AOS was constitutively expressed in different organs of flax plants. Additionally, AOS gene expression was enhanced after mechanical wounding in both the directly damaged leaves and in the systemic tissue located distal to the treated leaves. This wound-induced accumulation of AOS required the de novo biosynthesis of other unknown proteins involved in the signaling pathway modulating wound-induced AOS gene expression. Furthermore, the wound-induced AOS mRNA accumulation was correlated with the increase in the levels of JA. Both JA and its precursor, 12-oxo-phytodienoic acid, activated AOS gene expression in a dose-dependent manner. Thus, JA could activate its own biosynthetic pathway in flax leaves. Moreover, neither salicylic acid (SA) nor aspirin influenced AOS enzymatic activity. It is interesting that pretreatment with SA or aspirin inhibited wound-induced accumulation of AOS transcripts. These results suggest that a potent inhibition of JA biosynthetic capacity in leaves can be affected by SA or aspirin at the level of AOS gene expression.

Localized Wounding by Heat Initiates the Accumulation of Proteinase Inhibitor II in Abscisic Acid-Deficient Plants by Triggering Jasmonic Acid Biosynthesis.

To test whether the response to electrical current and heat treatment is due to the same signaling pathway that mediates mechanical wounding, we analyzed the effect of electric-current application and localized burning on proteinase inhibitor II (Pin2) gene expression in both wild-type and abscisic acid (ABA)-deficient tomato (Lycopersicon esculentum Mill.) and potato (Solanum phureja) plants. Electric-current application and localized burning led to the accumulation of Pin2 mRNA in potato and tomato wild-type plants. Among the treatments tested, only localized burning of the leaves led to an accumulation of Pin2 mRNA in the ABA-deficient plants. Electric-current application, like mechanical injury, was able to initiate ABA and jasmonic acid (JA) accumulation in wild-type but not in ABA-deficient plants. In contrast, heat treatment led to an accumulation of JA in both wild-type and ABA-deficient plants. Inhibition of JA biosynthesis by aspirin blocked the heat-induced Pin2 gene expression in tomato wild-type leaves. These results suggest that electric current, similar to mechanical wounding, requires the presence of ABA to induce Pin2 gene expression. Conversely, burning of the leaves activates Pin2 gene expression by directly triggering the biosynthesis of JA by an alternative pathway that is independent of endogenous ABA levels.

Expression of a Flax Allene Oxide Synthase cDNA Leads to Increased Endogenous Jasmonic Acid (JA) Levels in Transgenic Potato Plants but Not to a Corresponding Activation of JA-Responding Genes.

Both jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are thought to be significant components of the signaling pathway regulating the expression of plant defense genes in response to various stresses. JA and MeJA are plant lipid derivatives synthesized from [alpha]-linolenic acid by a lipoxygenase-mediated oxygenation leading to 13-hydroperoxylinolenic acid, which is subsequently transformed by the action of allene oxide synthase (AOS) and additional modification steps. AOS converts lipoxygenase-derived fatty acid hydroperoxide to allene epoxide, which is the precursor for JA formation. Overexpression of flax AOS cDNA under the regulation of the cauliflower mosaic virus 35S promoter in transgenic potato plants led to an increase in the endogenous level of JA. Transgenic plants had six- to 12-fold higher levels of JA than the nontransformed plants. Increased levels of JA have been observed when potato and tomato plants are mechanically wounded. Under these conditions, the proteinase inhibitor II (pin2) genes are expressed in the leaves. Despite the fact that the transgenic plants had levels of JA similar to those found in nontransgenic wounded plants, pin2 genes were not constitutively expressed in the leaves of these plants. Transgenic plants with increased levels of JA did not show changes in water state or in the expression of water stress-responsive genes. Furthermore, the transgenic plants overexpressing the flax AOS gene, and containing elevated levels of JA, responded to wounding or water stress by a further increase in JA and by activating the expression of either wound- or water stress-inducible genes. Protein gel blot analysis demonstrated that the flax-derived AOS protein accumulated in the chloroplasts of the transgenic plants.

Ac/Ds transposon mutagenesis in Arabidopsis thaliana: mutant spectrum and frequency of Ds insertion mutants.

Using a two-component Ac/Ds system consisting of a stabilized Ac element (Acc1) and a non-autonomous element (DsA), 650 families of plants carrying independent germinal DsA excisions/transpositions were isolated. Progenies of 559 of these Acc1/DsA families, together with 43 families of plants selected for excision/transposition of wild-type (wt) Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel DsA fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for DsA insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for DsA insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the DsA insertion could be separated from the mutant locus by genetic recombination.

Signals involved in wound-induced proteinase inhibitor II gene expression in tomato and potato plants.

Chemical and physical signals have been reported to mediate wound-induced proteinase inhibitor II (Pin2) gene expression in tomato and potato plants. Among the chemical signals, phytohormones such as abscisic acid (ABA) and jasmonic acid (JA) and the peptide systemin represent the best characterized systems. Furthermore, electrical and hydraulic mechanisms have also been postulated as putative Pin2-inducing systemic signals. Most of the chemical agents are able to induce Pin2 gene expression without any mechanical wounding. Thus, ABA, JA, and systemin initiate Pin2 mRNA accumulation in the directly treated leaves and in the nontreated leaves (systemic) that are located distal to the treated ones. ABA-deficient tomato and potato plants do not respond to wounding by accumulation of Pin2 mRNA, therefore providing a suitable model system for analysis of the signal transduction pathway involved in wound-induced gene activation. It was demonstrated that the site of action of JA is located downstream to the site of action of ABA. Moreover, systemin represents one of the initial steps in the signal transduction pathway regulating the wound response. Recently, it was reported that heat treatment and mechanical injury generate electrical signals, which propagate throughout the plant. These signals are capable of inducing Pin2 gene expression in the nontreated leaves of wounded plants. Furthermore, electrical current application to tomato leaves leads to an accumulation of Pin2 mRNA in local and systemic tissues. Examination of photosynthetic parameters (assimilation and transpiration rate) on several types of stimuli suggests that heat-induced Pin2 gene expression is regulated by an alternative pathway from that mediating the electrical current and mechanical wound response.

General roles of abscisic and jasmonic acids in gene activation as a result of mechanical wounding.

Exogenous application of abscisic acid (ABA) has been shown to induce a systemic pattern of proteinase inhibitor II (pin2) mRNA accumulation identical to that induced by mechanical wounding. Evidence is presented that the ABA-specific response is not restricted to pin2 genes but appears to be part of a general reaction to wound stress. Four other wound-induced, ABA-responsive genes that encode two additional proteinase inhibitors, the proteolytic enzyme leucine aminopeptidase, and the biosynthetic enzyme threonine deaminase were isolated from potato plants. Wounding or treatment with ABA resulted in a pattern of accumulation of these mRNAs very similar to that of pin2. ABA-deficient plants did not accumulate any of the mRNAs upon wounding, although they showed normal levels of expression upon ABA treatment. Also, application of methyl jasmonate (MeJA) induced a strong accumulation of these transcripts, both in wild-type and in ABA-deficient plants, thus supporting a role for jasmonic acid as an intermediate in the signaling pathway that leads from ABA accumulation in response to wounding to the transcriptional activation of the genes.

Factors affecting gene expression of patatin and proteinase-inhibitor-II gene families in detached potato leaves: : Implications for their co-expression in developing tubers.

In whole intact potato (Solanum tuberosum L.) plants, the gene families of class-I patatin and proteinase inhibitor II (Pin 2) are constitutively expressed in the tubers. However, they are also induced in detached potato leaves in the presence of light. To further characterize this light action, the detached leaves were subjected to monochromatic light of different wavelengths and to darkness in the presence of metabolites and inhibitors. Patatin genes could be induced by the simultaneous application of sucrose (sugars) and glutamine in darkness. Neither of these metabolites was active when supplied alone. When photosynthesis was blocked by 3-(3,4-Di-chlorophenyl)-1, 1-dimethylurea (DCMU) in the light, patatin genes were not expressed; however, the inhibition was overcome in the presence of sucrose. This indicates that besides its role in photosynthetic carbohydrate production, light may be essential for the supply of amino acids (or reduced nitrogen). Unlike patatin, Pin 2 genes were, to a small extent, also active in darkness, and sucrose weakly enhanced this expression. However, DCMU did not affect Pin 2 expression in the light. Both abscisic acid and methyl jasmonate strongly promoted the accumulation of Pin 2 mRNA independent of the light conditions, indicating that the gene family is probably under hormonal control. The phytohormones did not affect patatin gene expression. Inhibitors of cytosolic (cycloheximide) and organellar (chloramphenicol) translation had opposite effects on the two gene families. Careful evaluation of the inhibitors' action indicates that protein synthesis (cytosol) is required for the expression of Pin 2 genes but not for the patatin genes. These results clearly demonstrate that, although in situ both gene families are constitutively expressed in the same plant organ (tuber) in intact plants, their expression is mediated by different factors.

Abscisic Acid Mediates Wound Induction but Not Developmental-Specific Expression of the Proteinase Inhibitor II Gene Family.

The expression of the potato and tomato proteinase inhibitor II (pin2) gene family is subject to both developmental and environmental control, being constitutively expressed in potato tubers while only being present in the foliage of the potato or tomato plants after mechanical damage. There is evidence that the phytohormone abscisic acid (ABA) is involved in this wound induction of pin2 gene expression. This paper describes experiments that demonstrate that ABA is able to induce the expression of the pin2 gene family, both locally and systemically, at physiological concentrations. The significance of the ABA involvement in the pin2 induction upon wounding has been further strengthened by analyzing the expression of a pin2 promoter-[beta]-glucuronidase gene fusion in transgenic ABA-deficient mutant potato plants. We have analyzed the developmental regulation of pin2 gene expression in wild-type and ABA-deficient potato and tomato plants. The pin2 mRNA level is identical in mutant and wild-type parental Solanum phureja tubers. In addition, evidence is presented for pin2 also being constitutively expressed at certain stages in the development of both tomato and potato flowers. Again, the ABA deficiency appears to have little influence in this tissue-specific expression in the mutants. These results suggest the action of separate pathways for the developmental and environmental regulation of pin2 gene expression.

Sagaxine, the protamine from Sardinops sagax. Characterization, sequence and secondary structure prediction.

This paper describes the isolation, purification and full characterization of the protamine from Sardinops sagax. The protein was purified by both gel filtration and ion exchange chromatography and its amino acid composition, electrophoretic behaviour and sequence were determined. We postulate that the protamine exhibits microheterogeneity with a major and a minor component. The molecular weight for the major component is 4377, as calculated from the sequence. The N-terminal amino acid is proline and the C-terminal is arginine. The amino acid composition is: Thr-Ser-Glx-(Pro)2-Gly-(Ala)3-(Val)2-(Lys)2-(Arg)20 The secondary structure for the protamines was also predicted and a revision of our previously published results in this area is discussed. We have called this protamine Sagaxine and an overall comparison with the hitherto described fish protamines is also made.

Identification of potato nuclear proteins binding to the distal promoter region of the proteinase inhibitor II gene.

Potato nuclear proteins specifically bind to a DNA sequence at the most 5' distal region of the promoter of a potato proteinase inhibitor II gene. Binding studies using the electrophoretic mobility-shift assay showed the appearance of two protein-DNA complexes in the presence of both tuber and leaf nuclear protein extracts. Mechanical wounding of the leaves had no effect on the amount of specific protein-DNA complexes formed. DNase I protection analysis and binding to synthetic oligonucleotides identified the sequence 5'-GAGGGTATTTTCGTAA-3' as the target for the noncooperative binding of two potato nuclear proteins to the upstream element. Methylation interference experiments showed that guanine nucleotides separated by one turn of the DNA helix were in close contact with the proteins. The binding ability of a series of mutated synthetic oligonucleotides further defined the sequence requirements for protein binding, which appeared to contact one side of the DNA helix.

Gene expression during tuber development in potato plants.

Potato tubers are modified stems that have differentiated into storage organs. Factors such as day-length, nitrogen supply, and levels of the phytohormones cytokinin and gibberellic acid, are known to control tuberization. Morphological changes during tuber initiation are accompanied by the accumulation of a characteristic set of proteins, thought to be involved in N-storage (i.e. patatin) or defense against microbial or insect attack (i.e. proteinase inhibitor II). Additionally, deposition of large amounts of starch occurs during tuber formation, which is paralleled by an increase in sucrose synthase and other enzymes involved in starch biosynthesis (i.e. ADP-glucose pyrophosphorylase, starch synthases, and branching enzyme). Potential controlling mechanisms for genes expressed during tuberization are discussed.

Abscisic acid is involved in the wound-induced expression of the proteinase inhibitor II gene in potato and tomato.

Plants respond to wounding or pathogen attack by a variety of biochemical reactions, involving in some instances gene activation in tissues far apart from the actual site of wounding or pathogen invasion. One of the best analyzed examples for such a systemic reaction is the wound-induced expression of proteinase inhibitor genes in tomato and potato leaves. Local wounding of potato or tomato plants results in the accumulation of proteinase inhibitors I and II throughout the aerial part of the plant. In contrast to wild-type plants, abscisic acid-deficient mutants of potato (droopy) and tomato (sit) show a drastically reduced induction of these genes in response to plant wounding. High levels of proteinase inhibitor II gene expression are obtained in mutant and wild-type plants upon exogenous application of abscisic acid. Measurements of the endogenous abscisic acid levels in wild-type plants show that wounding results in increased levels of this phytohormone in wounded and nonwounded systemically induced leaves. Thus these results show that the plant hormone abscisic acid is involved in the wound-induced activation of the proteinase inhibitor II gene. Furthermore, they are compatible with a model assuming this hormone to be the actual mediator of the systemic wound response.

Systemic induction of proteinase-inhibitor-II gene expression in potato plants by wounding.

The systemic induction of expression of the gene for proteinase inhibitor II after wounding different parts of potato (Solanum tuberosum L.) plants was analysed at the RNA level. Wounding of either leaves or tubers led to an induction of expression of this gene in non-wounded upper and lower leaves as well as in the upper stem segment, whereas no expression was observed in nonwounded roots or in the lower stem segment. The signal mediating the systemic induction in nonwounded tissue must therefore be able to move both acropetally and basipetally. The systemic wound response is specific for the expression of the proteinase-inhibitor-II gene as no influence was observed for the expression of genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the tuber storage protein patatin which were examined in parallel with the proteinase-inhibitor-II gene.