Relationship between H2O2 and jasmonic acid in pea leaf wounding response

The relationship of H₂O₂ and jasmonic acid (JA) in wound-induced defense response was investigated in the leaves of pea (Pisum sativum L.) plants. The results showed that both wounding and JA treatment led to a significant increase in activities of plasma membrane NADPH oxidase and phenylalanine ammonialyase. However, such an increase was blocked by the pretreatment with plasma membrane NADPH oxidase inhibitors, O ₂ ^? scavengers, or H₂O₂ scavenger, implying that H₂O₂ functions downstream of JA. Furthermore, wounding treatment activated two key enzymes of JA biosynthesis, lipoxygenase and allene oxide synthase, while JA biosynthetic inhibitors impaired the wounding-induced H₂O₂ burst. Thus, it is suggested that H₂O₂ burst depends on JA production in plant wounding response.     

Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress,wounding, and salicylic acid treatment in Carthamus tinctorius

Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3–6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6–24 h and 3–6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance.     

Differential expression of two cinnamate 4-hydroxylase genes in 'Valencia' orange (Citrus sinensis Osbeck)

Two different full-length cDNAs for cinnamate 4-hydroxylase (C4H1 and C4H2) were isolated from Citrus sinensis Osbeck cv. Valencia libraries. C4H1 (1708 bp) and C4H2 (1871 bp) share only 65% identity on nucleotide and 66% identity on the amino acid level, respectively. C4H1 is most homologous to a cinnamate 4-hydroxylase sequence from French bean (Phaseolus vulgaris), but codes for a unique N-terminus. C4H2 shows highest similarity to a poplar (Populus kitakamiensis) sequence, but also shows a unique N-terminus. The two genes are expressed differentially in orange flavedo, C4H2 is constitutive, C4H1 is wound-induced. In competitive RT-PCR, the mRNA for both genes in wounded and untreated tissue was quantified. C4H1 is strongly wound-inducible from `not detectable' to about 35 fg mRNA per 50 ng total RNA 8 h after wounding. The first detectable C4H1 mRNA was found 4 h after wounding. After reaching peak levels 4 h later the levels slightly declined, but stayed elevated until the end of the experiment (48 h). C4H2 is expressed 3–10 times higher than wound-induced C4H1 even in the control sample; wounding transiently increases the level of expression another 2–3 times. The existence of different N-termini and their effects on the possible role of both genes in phenylpropanoid pathways is discussed.     

Multiple cis-regulatory elements regulate distinct and complex patterns of developmental and wound-induced expression of Arabidopsis thaliana 4CL gene family members

Lignin is an important biopolymer that is deposited in secondary cell walls of plant cells (e.g., tracheary elements) and in response to stresses such as wounding. Biosynthesis of lignin monomers occurs via the phenylpropanoid pathway, in which the enzyme 4-coumarate:CoA ligase (4CL) plays a key role by catalyzing the formation of hydroxycinnamoyl-CoA esters, subsequently reduced to the corresponding monolignols (hydroxycinnamoyl alcohols). 4CL is encoded by a family of four genes in Arabidopsis thaliana (At4CL1-At4CL4), which are developmentally regulated and co-expressed with other phenylpropanoid genes. We investigated in detail the wound-induced expression of At4CL1-At4CL4, and found that At4CL1 and At4CL2 mRNA accumulation follows biphasic kinetics over a period of 72 h, while At4CL4 expression is rapidly activated for a period of at least 12 h before declining. In order to localize cis-regulatory elements involved in the developmental and wound-induced regulation of the At4CL gene family members, At4CL promoter-beta-glucuronidase (GUS) reporter gene fusions were constructed and transferred into Arabidopsis plants. Analysis of these plants revealed that the promoter fragments direct discrete and distinct patterns of expression, some of which did not recapitulate expected patterns of wound-induced expression. The locations of regulatory elements associated with the At4CL2 gene were investigated in detail using a series of transgenic Arabidopsis plants containing promoter fragments and parts of the transcribed region of the gene fused to GUS. Positive and negative regulatory elements effective in modulating developmental expression or wound responsiveness of the gene were located both in the promoter and transcribed regions of the At4CL2 gene.An erratum to this article can be found at     

Systemic induction of H2O2 in pea seedlings pretreated by wounding and exogenous jasmonic acid

Pea seedlings (Pisum sativum L.) were used as materials to test the timings and compartments of hydrogen peroxide (H₂O₂) triggered by wounding and exogenous jasmonic acid (JA). The results showed that H₂O₂ could be systemically induced by wounding and exogenous JA. H₂O₂ increased within 1 h and reached the peak 3–5 h after wounding in either the wounded leaves or the unwounded leaves adjacent to the wounded ones and the inferior leaves far from the wounded ones. After this, H₂O₂ decreased and recovered to the control level 12 h after wounding. The activities of antioxidant enzymes, however, were rapidly increased by wounding. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, could significantly inhibit H₂O₂ burst that was mediated by wounding and exogenous JA. Assay of H₂O₂ subcellular location showed that H₂O₂ in response to wounding and exogenous JA was predominantly accumulated in plasma membrane, cell wall and apoplasmic space. Numerous JA (gold particles) was found via immunogold electron microscopy to be located in cell wall and phloem zones of mesophyll cell after wounding.     

Wounding of potato tubers induces increases in ABA biosynthesis and catabolism and alters expression of ABA metabolic genes

The effects of physical wounding on ABA biosynthesis and catabolism and expression of genes encoding key ABA metabolic enzymes were determined in potato tubers. An increase in ABA and ABA metabolite content was observed 48 h after wounding and remained elevated through 96 h. Wounding induced dramatic increases in the expression of the ABA metabolic genes encoding zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), and ABA-8′-hydroxylase. Although the patterns of wound-induced expression of individual genes varied, increased gene expression was observed within 3 h of wounding and remained elevated through 96 h. An apparent correlation between expression of the gene encoding ZEP and the increase in ABA content suggested that the wound-induced increase in ABA biosynthesis was regulated by both substrate availability and increased NCED activity. Suppression of wound-induced jasmonic acid accumulation by rinsing the wounded tissue with water did not inhibit the subsequent increase in ABA content. Exogenous ethylene completely suppressed the wound-induced increase in ABA content and dramatically reduced wound-induced up-regulation of ABA metabolic genes. This study is the first to identify the molecular bases for increased ABA accumulation following physical trauma in potato tubers and highlights the complex physiological interactions between various wound-induced hormones.     

Differential expression profiles of poplar MAP kinase kinases in response to abiotic stresses and plant hormones,and overexpression of PtMKK4 improves the drought tolerance of poplar

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play essential roles in plant growth, development and stress response. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), are integral in mediating various stress responses in plants. However, to date few data about the roles of poplar MAPKKs in stress signal transduction are available. In this study, we performed a systemic analysis of poplar MAPKK gene family expression profiles in response to several abiotic stresses and stress-associated hormones. Furthermore, Populus trichocarpa MAPKK4 (PtMKK4) was chosen for functional characterization. Transgenic analysis showed that overexpression of the PtMKK4 gene remarkably enhanced drought stress tolerance in the transgenic poplar plants. The PtMKK4-overexpressing plants also exhibited much lower levels of H₂O₂ and higher antioxidant enzyme activity after exposure to drought stress compared to the wide type lines. Besides, some drought marker genes including PtP5CS, PtSUS3, PtLTP3 and PtDREB8 exhibited higher expression levels in the transgenic lines than in the wide type under drought conditions. This study provided valuable information for understanding the putative functions of poplar MAPKKs involved in important signaling pathways under different stress conditions.     

Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan-3-ols) accumulation in tea

Phenylalanine ammonia-lyase and cinnamate 4-hydroxylase are important enzymes in allocating significant amounts of carbon from phenylalanine into the biosynthesis of several important secondary metabolites. Tea is an important crop of commerce known for its beverage and medicinally important flavonoid compounds, mainly catechins. As metabolic flux for the operation of the flavonoid pathway is maintained through the activities of PAL and C4H, thus, catechins biosynthesis in tea is critically dependent on the products of these enzymes. We examined the expression of PAL and C4H. Sequence encoding CsPAL was isolated from tea by polymerase chain reaction using sequence information available at the NCBI GenBank. Sequence encoding C4H was isolated from tea by using differential display of mRNA and rapid amplification of cDNA ends technology. CsC4H (AY641731) comprised of 1,352 bp full-length cDNA with open reading frame of 1,173 bp encoding 390 amino acids. Catechin contents decreased in response to drought stress (DS), abscisic acid (ABA), and gibberellic acid (GA₃) treatments but increased in response to wounding. The expression of CsPAL and CsC4H showed the same behavior under the above treatments and was also in accordance with the catechin contents. A positive correlation between catechin contents and gene expression suggested a critical role of the enzymes in catechins biosynthesis and a crosstalk between phenylpropanoid and flavonoid pathways.     

Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding,salicylic acid treatment,and salinity stress in safflower,Carthamus tinctorius

Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT–PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress.     

Flower colour and cytochromes P450

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.     

Reduced levels of volatile emissions in jasmonate-deficient spr2 tomato mutants favour oviposition by insect herbivores

Tomato plants release volatile organic compounds (VOCs) following insect or mechanical damage. In this study, the constitutive and wound-induced emission levels of VOCs in suppressor of prosystemin-mediated responses2 (spr2) mutant plants, compromised in linolenic acid (LA) and jasmonic acid (JA) synthesis, and in 35S::prosystemin (35S::prosys) plants, having upregulated direct defence responses, were compared. The spr2 mutants produced constitutively lower levels of VOCs, which were nonetheless increased in response to (a)biotic damage, although at lower levels than wild-type (Wt) and 35S::prosys plants. No significant differences in VOC emissions were detected between the latter two genotypes, thereby suggesting that systemin does not regulate indirect defence responses, whereas differences in fatty acid composition in spr2 plants led to the predominant emission of saturated C6 volatiles in response to wounding. The expression of 1-deoxy-D-xylulose 5-phosphate synthase (DXS2), a key gene involved in VOC synthesis in the chloroplast, was only upregulated in Manduca sexta L.-damaged Wt and 35S::prosys plants. However, its expression was restored in spr2 plants by exogenous LA or JA, suggesting that abated VOC emissions in spr2 plants are correlated with lowered DXS2 expression. Bioassays with two different insects showed that adult females significantly preferred spr2 plants, indicating that lowered levels of VOCs in tomato influence plant selection by insects during oviposition.     

Exogenous caffeic acid inhibits the growth and enhances the lignification of the roots of soybean (Glycine max)

The allelopathic effect of caffeic acid was tested on root growth, phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, hydrogen peroxide (H₂O₂) accumulation, lignin content and monomeric composition of soybean (Glycine max) roots. We found that exogenously applied caffeic acid inhibited root growth, decreased the PAL activity and H₂O₂ content and increased the soluble and cell wall-bound POD activities. The p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monomers and total lignin (H + G + S) increased in the caffeic acid-exposed roots. When applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), caffeic acid equalized the inhibitory effect of PIP, whereas the application of methylene dioxocinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL) plus caffeic acid decreased lignin production. These results indicate that exogenously applied caffeic acid can be channeled into the phenylpropanoid pathway via the 4CL reaction, resulting in an increase of lignin monomers that solidify the cell wall and inhibit root growth.     

Coordinate expression of AOS genes and JA accumulation: JA is not required for initiation of closing layer in wound healing tubers

Wounding induces a series of coordinated physiological responses essential for protection and healing of the damaged tissue. Wound-induced formation of jasmonic acid (JA) is important in defense responses in leaves, but comparatively little is known about the induction of JA biosynthesis and its role(s) in tuber wound-healing. In this study, the effects of wounding on JA content, expression of JA biosynthetic genes, and the involvement of JA in the initiation of closing layer formation in potato tubers were determined. In addition, the role of abscisic acid (ABA) in wound-induced JA accumulation was examined. The basal JA content in non-wounded tuber tissues was low (<3 ng g⁻¹ FW). Two hours after wounding, the JA content increased by >5-fold, reached a maximum between 4 and 6 h after wounding, and declined to near-basal levels thereafter. Tuber age (storage duration) had little effect on the pattern of JA accumulation. The expressions of the JA biosynthetic genes (StAOS2, StAOC, and StOPR3) were greatly increased by wounding reaching a maximum 2-4 h after wounding and declining thereafter. A 1-h aqueous wash of tuber discs immediately after wounding resulted in a 94% inhibition of wound-induced JA accumulation. Neither JA treatment nor inhibition of JA accumulation affected suberin polyphenolic accumulation during closing layer development indicating that JA was not essential for the initiation of primary suberization. ABA treatment did not restore JA accumulation in washed tuber tissues suggesting that leaching of endogenous ABA was either not involved or not solely involved in this loss of JA accumulation by washing. Collectively, these results indicate that JA is not required for the induction of processes essential to the initiation of suberization during closing layer development, but do not exclude the possibility that JA may be involved in other wound related responses.