Abscisic acid-deficient plants do not accumulate proteinase inhibitor II following systemin treatment

The role of systemin inPin2 gene expression was analyzed in wild-type plants of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum Mill.), as well as in abscisic acid (ABA)-deficient tomato (sitiens) and potato (droopy) plants. The results showed that systemin initiates Pin2 mRNA accumulation only in wildtype tomato and potato plants. As in the situation after mechanical wounding,Pin2 gene expression in ABA-deficient plants was not activated by systemin. Increased endogenous levels of jasmonic acid (JA) and accumulation of Pin2 mRNA were observed following treatment with α-linolenic acid, the precursor of JA biosynthesis, suggesting that these ABA mutants still have the capability to synthesize de novo JA. Measurement of endogenous levels of ABA and JA showed that systemin leads to an increase of both phytohormones (ABA and JA) only in wild-type but not in ABA-deficient plants.     

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.The plant hormones ABA and JA play a predominant role in the conversion of environmental signals into changes in plant gene expression. An increase in endogenous ABA and JA levels precedes and is involved in Pin2 (proteinase inhibitor II) gene expression upon wounding (Peña-Cortés et al., 1989, 1991, 1995, 1996; Farmer and Ryan, 1992; Farmer et al., 1992). This increase in ABA and JA is not restricted to the tissue damaged directly but can also be detected in the nonwounded, systemically induced tissue (Peña-Cortés et al., 1989; Peña-Cortés and Willmitzer, 1995). The accumulation of ABA and JA have been described for several plant species, including potato, tomato, and tobacco (Sanchez-Serrano et al., 1991; Peña-Cortés and Willmitzer, 1995).Further evidence for the involvement of ABA and JA in wound-induced Pin2 gene expression was provided by a series of experiments in which potato plants were sprayed with ABA or JA and Pin2 mRNA accumulated in the absence of any wounding (Peña-Cortés et al., 1989; Hildmann et al., 1992). Both nonsprayed leaves and leaves that were sprayed directly showed increased Pin2 mRNA levels with a pattern identical to the one described for wounded plants (Peña-Cortés et al., 1988; Peña-Cortés and Willmitzer, 1995). Conclusive evidence for the involvement of ABA in wound-induced Pin2 activation was obtained from mutants impaired in ABA biosynthesis. Consequently, wound induction of Pin2 was not observed in the droopy mutant of potato or the sitiens mutant of tomato (Peña-Cortés et al., 1989). However, in these mutants treatment with ABA caused a return of the accumulation of Pin2 mRNA to levels normally found in wild-type plants upon wounding (Peña-Cortés et al., 1991).Like wounding, the application of electrical current was able to initiate ABA and JA accumulation in wild-type plants but not in ABA-deficient plants (Herde et al., 1996). These results suggested that, like wounding, electrical current requires the presence of ABA for the induction of Pin2 gene expression (Herde et al., 1996). In contrast to wounding and electrical current, burning of leaves activated Pin2 gene expression in sitiens mutants by directly triggering the biosynthesis of JA via an alternative pathway that is independent of endogenous ABA levels (Herde et al., 1996).To determine the endogenous levels of ABA that are sufficient to mediate electrical current-, heat-, and wound- induced Pin2 gene expression via electrical signals, we used several tomato mutants containing progressively reduced levels of ABA. The effects of these attenuated ABA levels on JA content and membrane potentials and the expression pattern of Pin2 genes were analyzed. Analysis of JA content was conducted to confirm the existence of an alternative pathway that is independent of endogenous ABA levels in the different ABA-deficient mutants.     

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.     

Time-Resolved Analysis of Signals Involved in Systemic Induction of Pin2 Gene Expression

The systemic induction of proteinase inhibitor genes in tomato plants is mediated either by electrical signals, hydraulic signals or chemical messengers. In the present study we analyzed the effects of mechanical wounding, heat treatment and electrical current application on wild-type tomato plants (Lycopersicon esculentum Mill, cv Moneymaker) and ABA-deficient mutants of tomato (sitiens). Kinetic studies revealed that systemic Pin2 gene expression could be slightly induced by the fast transient membrane potential change which left the damaged leaf within 30–60s after wounding. Moreover, a signal leaving the damaged tissue between 2 and 4 minutes after wounding was responsible for a significant amplification of Pin2 gene expression. This signal could either be a decrease in turgor pressure, which occurred 3–4min after treatment, or a slow electrical transient. In addition, mechanical wounding and electrical current seem to involve ABA to induce changes in membrane potential and to promote Pin2 gene expression. In contrast, heat triggers fast and slow electrical transients leading to an induction of Pin2 gene expression within the plant independently of ABA. Turgor pressure, in turn, is presumably adjusted in relation to ionic movements across the membrane, elucidated by membrane potential recordings. In conclusion, wound-induced changes in membrane potential seem to be dependent on the endogenous level of ABA. These shifts in membrane potentials, in turn, are involved in regulation of turgor pressure within the plant.     

Aspirin prevents wound-induced gene expression in tomato leaves by blocking jasmonic acid biosynthesis

Jasmonic acid (JA) and its methyl ester, like mechanical wounding, strongly induce accumulation of proteinase inhibitor II (Pin2) in tomato and potato leaves. In plants, JA is synthesized from α-linolenic acid by a lipoxygenase (LOX)-mediated oxygenation leading to 13-hydroxyperoxylinolenic acid (13-HPLA) which is then subsequently transformed to JA by the action of hydroperoxide-dehydrase activity and additional modification steps. Both the chemical structure as well as the biosynthetic pathway of JA resemble those of the mammalian eicosanoids (prostaglandins and leukotrienes) which are derived from LOX-and cyclooxygenase (COX)-mediated reactions. To assess the role of endogenous JA in the wound response, detached tomato (Lycopersicon esculentum Mill.) leaves were supplied with different LOX and COX inhibitors and the expression of the wound-induced genes for Pin2 (Pin2), cathepsin D inhibitor (Cdi) and threonine deaminase (Td) was analyzed. Lipoxygenase inhibitors as well as some COX inhibitors blocked the wound-induced accumulation of Pin2, Cdi and Td mRNA. Quantitation of endogenous levels of JA showed that aspirin blocks the increase of this phytohormone normally observed as a result of wounding. Linolenic acid and 13-HPLA do not induce the expression of Pin2, Cdi and Td in the presence of aspirin. However, 12-oxo-phytodienoic acid and jasmonic acid are able to overcome the inhibitory effect of this substance. These results strongly indicate that aspirin prevents wound-induced gene activation by inhibiting the hydroxyperoxide-dehydrase activity that mediates the conversion of 13-HPLA to 12-oxo-phytodienoic acid.     

机械伤害诱导的植物防御机制和信号转导

茉莉酸是植物伤反应的特异激素, 在植物伤反应中具有核心作用, 其下游调控机制已经比较清晰。在番茄(Lycopersicon esculentum)伤反应中, 系统素和茉莉酸协同启动相关基因的表达, 行使系统性防御功能。拟南芥(Arabidopsis thaliana)信号肽是新发现的一类信号物质, 可以激活植物的初始免疫反应, 但其在伤反应中的作用机制有待进一步研究。脱落酸位于茉莉酸上游, 单独或者协同茉莉酸参与植物的防御反应。另外, 植物中还存在以核糖核酸酶为代表的且不依赖于茉莉酸的伤反应信号转导途径。该文对植物伤反应的防御机制和信号转导做了详细概述。     

Proteinase Inhibitor II Gene Expression Induced by Electrical Stimulation and Control of Photosynthetic Activity in Tomato Plants

Mechanical damage and heat stimulation were used to activateproteinase inhibitor II (Pin2) gene expression in tomato plantsin both treated (local induction) and non-treated tissues (systemicinduction). Both stimuli have been shown to generate electricalsignals, leading to a systemic activation of gene expression.Treatment of tomato leaves with electrical current resultedin the accumulation of Pin2 mRNA in the local and systemic leaves.Additionally, all treatments inducing Pin2 gene activity gaverise to a significant alteration of stomatal aperture. However,heat stimulation provoked a different response in the stomatalparameters than mechanical wounding or electric treatment. Bothmechanical damage and electrical stimulation activated two characteristictime constants in the gas exchange relaxation kinetics. Conversely,heat stimulation resulted in only one major time constant. Theresults clearly show that direct current application to tomatoleaves initiates Pin2 mRNA accumulation locally and systemically.In addition, they suggest the participation of a second slowelectrical/hydraulic component in the wound response mechanismof tomato plants and a possible alternative pathway regulatingheat-induced Pin2 gene expression. (Received February 13, 1995; Accepted April 14, 1995)     

Abscisic acid and jasmonic acid activate wound-inducible genes in potato through separate, organ-specific signal transduction pathways

Mechanical damage to leaf tissue causes an increase in abscisic acid (ABA) which in turn activates the biosynthesis of jasmonic acid (JA). The resulting higher endogenous JA levels subsequently activate the expression of wound-inducible genes. This study shows that JA induces the expression of different sets of genes in roots and leaves of potato plants. When roots of intact plants were treated with JA, high levels of proteinase inhibitor II (pin2), cathepsin D inhibitor, leucine aminopeptidase and threonine deaminase mRNAs accumulated in the systemic leaves. However, in the treated roots, very low, if any, expression of these genes could be detected. In contrast, a novel, root-specific pin2 homologue accumulated in the JA-treated root tissue which could not be detected in leaves, either systemic or those directly treated with JA. Application of okadaic acid and staurosporine revealed that a protein phosphorylation step is involved in the regulation of this differential response. In leaves, a protein phosphatase is required for the JA-induced expression of pin2 and the other genes analysed. This phosphatase activity is not necessary for the JA-induced expression of a pin2 homologue in roots, suggesting the existence of different transduction pathways for the JA signal in these organs. The requirement of a protein phosphatase activity for JA-mediated gene induction has enabled identification of a JA-independent pathway for ABA induction of pin2 and the other wound-inducible genes. This alternative pathway involves a protein kinase, and appears to be selective for wound-inducible genes. Our data suggest the presence of a complex, organ-specific transduction network for regulating the effects of the plant hormones ABA and JA on gene expression upon wounding.     

Stomatal responses to jasmonic acid, linolenic acid and abscisic acid in wild-type and ABA-deficient tomato plants

Wild-type and abscisic acid (ABA) -deficient (sitiens) tomato plants were used to analyse the effects of abscisic acid (ABA), butyric acid (BA), jasmonic acid (JA) and linolenic acid (LA) on assimilation and transpiration rates in detached leaves taking up those substances into the transpiration stream. BA did not affect assimilation and transpiration rates. ABA decreased assimilation and transpiration in both wild-type and ABA-deficient mutants. JA reduced the assimilation rate in both lines but induced a significant reduction of transpiration in the wild type only. The response to LA in both lines was slower than that to JA.     

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.     

Expression of a GALACTINOL SYNTHASE gene in tomato seeds is up-regulated before maturation desiccation and again after imbibition whenever radicle protrusion is prevented

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance.     

Induction of the Arabidopsis PHO1;H10 gene by 12-oxo-phytodienoic acid but not jasmonic acid via a CORONATINE INSENSITIVE1-dependent pathway

Expression of AtPHO1;H10, a member of the Arabidopsis (Arabidopsis thaliana) PHO1 gene family, is strongly induced following numerous abiotic and biotic stresses, including wounding, dehydration, cold, salt, and pathogen attack. AtPHO1;H10 expression by wounding was localized to the cells in the close vicinity of the wound site. AtPHO1;H10 expression was increased by application of the jasmonic acid (JA) precursor 12-oxo-phytodienoic acid (OPDA), but not by JA or coronatine. Surprisingly, induction of AtPHO1;H10 by OPDA was dependent on the presence of CORONATINE INSENSITIVE1 (COI1). The induction of AtPHO1;H10 expression by wounding and dehydration was dependent on COI1 and was comparable in both the wild type and the OPDA reductase 3-deficient (opr3) mutant. In contrast, induction of AtPHO1;H10 expression by exogenous abscisic acid (ABA) was independent of the presence of either OPDA or COI1, but was strongly decreased in the ABA-insensitive mutant abi1-1. The involvement of the ABA pathway in regulating AtPHO1;H10 was distinct between wounding and dehydration, with induction of AtPHO1;H10 by wounding being comparable to wild type in the ABA-deficient mutant aba1-3 and abi1-1, whereas a strong reduction in AtPHO1;H10 expression occurred in aba1-3 and abi1-1 following dehydration. Together, these results reveal that OPDA can modulate gene expression via COI1 in a manner distinct from JA, and independently from ABA. Furthermore, the implication of the ABA pathway in coregulating AtPHO1;H10 expression is dependent on the abiotic stress applied, being weak under wounding but strong upon dehydration.     

Allene oxide cyclase dependence of the wound response and vascular bundle-specific generation of jasmonates in tomato - amplification in wound signalling

The allene oxide cyclase (AOC)-catalyzed step in jasmonate (JA) biosynthesis is important in the wound response of tomato. As shown by treatments with systemin and its inactive analog, and by analysis of 35S::prosysteminsense and 35S::prosysteminantisense plants, the AOC seems to be activated by systemin (and JA) leading to elevated formation of JA. Data are presented on the local wound response following activation of AOC and generation of JA, both in vascular bundles. The tissue-specific occurrence of AOC protein and generation of JA is kept upon wounding or other stresses, but is compromised in 35S::AOCsense plants, whereas 35S::AOCantisense plants exhibited residual AOC expression, a less than 10% rise in JA, and no detectable expression of wound response genes. The (i). activation of systemin-dependent AOC and JA biosynthesis occurring only upon substrate generation, (ii). the tissue-specific occurrence of AOC in vascular bundles, where the prosystemin gene is expressed, and (iii). the tissue-specific generation of JA suggest an amplification in the wound response of tomato leaves allowing local and rapid defense responses.     

Wild-type levels of abscisic Acid are not required for heat shock protein accumulation in tomato

Levels of endogenous abscisic acid (ABA) in wild type were not required for the synthesis of heat shock proteins in detached leaves of tomato (Lycopersicon esculentum Mill., cv Ailsa Craig). Heat-induced alterations in gene expression were the same in the ABA-deficient mutant of tomato, flacca, and the wild type. Heat tolerance of the mutant was marginally less that the wild type, and in contrast, ABA applications significantly reduced the heat tolerance of wild-type leaves. It was concluded that elevated levels of endogenous ABA are not involved in the tomato heat shock response.