Stress responses in alfalfa (Medicago sativa L.) III. Induction of medicarpin and cytochrome P450 enzyme activities in elicitor-treated cell suspension cultures and protoplasts

Summary Cell suspension cultures of alfalfa (Medicago sativa L.) accumulated phenolic secondary metabolites in a pattern similar to that seen in alfalfa roots. Upon treatment with a crude elicitor preparation from the bean pathogen Colletotrichum lindemuthianum, the pterocarpan phytoalexin medicarpin accumulated in cells and culture medium. The extractable activities of six enzymes involved in medicarpin biosynthesis (including three cytochrome P450 activities) were induced by treatment with elicitor, and their induction kinetics correlated with the rate of medicarpin accumulation. However, protoplasts prepared from these cultures accumulated neither medicarpin nor other secondary products after treatment with elicitor. The cytochrome P450 activities were induced during the preparation of the protoplasts, but could be further induced by treatment with fungal elicitor. The results are discussed in relation to the use of alfalfa protoplasts as a system for functional analysis of cloned defense genes.Abbreviations AUFS absorption unit full scale - CHI chalcone isomerase (EC 5.5.1.6) - CHS chalcone synthase (EC 2.3.1.74) - C40H cinnamic acid 4-hydroxylase (EC 1.14.13.11) - CLE elicitor from Colletotrichum lindemuthianum - IFOH isoflavone 2-hydroxylase - IFS isoflavone synthase - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Cloning of soybean genes induced during hypersensitive cell death caused by syringolide elicitor

Syringolide elicitors produced by bacteria expressing Pseudomonas syringae pv. glycinea avirulence gene D (avrD) induce hypersensitive cell death (HCD) only in soybean (Glycine max [L.] Merr.) plants carrying the Rpg4 disease resistance gene. Employing a differential display method, we isolated 13 gene fragments induced in cultured cells of a soybean cultivar Harosoy (Rpg4) treated with syringolides. Several genes for isolated fragments were induced by syringolides in an rpg4 cultivar Acme as well as in Harosoy; however, the genes for seven fragments designated as SIH (for syringolide-induced/HCD associated) were induced exclusively or strongly in Harosoy. cDNA clones for SIH genes were obtained from a cDNA library of Harosoy treated with syringolide. Several sequences are homologous to proteins associated with plant defense responses. The SIH genes did not respond to a non-specific -glucan elicitor, which induces phytoalexin accumulation but not HCD, suggesting that the induction of the SIH genes is specific for the syringolide–Harosoy interaction. HCD and the induction of SIH genes by syringolides were independent of H₂O₂. On the other hand, Ca²⁺ was required for HCD and the induction of some SIH genes. These results suggest that the induction of SIH genes by syringolides could be activated through the syringolide-specific signaling pathway and the SIH gene products may play an important role(s) in the processes of HCD induced by syringolides.Abbreviations AOS active oxygen species - CHS chalcone synthase - DPI diphenylene iodonium - HCD hypersensitive cell death - HR hypersensitive response - PAL phenylalanine ammonia lyase - SID syringolide-induced/defense associated - SIG syringolide-induced/general - SIH for syringolide-induced/HCD associated - XET xyloglucan endotransglycosylase     

Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.)

Genes involved in flavonoid and stilbene biosynthesis were isolated from grape (Vitis vinifera L.). Clones coding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydoxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), were isolated by screening a cDNA library, obtained from mRNA from seedlings grown in light for 48 h using snapdragon (Antirrhinum majus) and maize heterologous probes. A cDNA clone coding for stilbene synthase (StSy) was isolated by probing the library with a specific oligonucleotide. These clones were sequenced and when the putative products were compared to the published amino acid sequence for corresponding enzymes, the percentages of similarity ranged from 65% (UFGT) to 90% (CHS and PAL). The analysis of the genomic organization and expression of these genes in response to light shows that PAL and StSy genes belong to large multigene families, while the others are present in one to four copies per haploid genome. The steady-state level of mRNAs encoded by the flavonoid biosynthetic genes as determined in young seedlings is coordinately induced by light, except for PAL and StSy, which appear to be constitutively expressed.     

Elicitor-induced metabolic changes in cell cultures of chickpea (Cicer arietinum L.) cultivars resistant and susceptible to Ascochyta rabiei

Cell-suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant (ILC 3279) and susceptible (ILC 1929) to the fungus Ascochyta rabiei (Pass.) Lab., showed differential accumulation of the phytoalexins medicarpin and maackiain, and transient induction of related enzyme activities after application of an A. rabiei-derived elicitor. The chalcone-synthase (CHS) activity (EC 2.3.1.74) which is involved in the first part of phytoalexin biosynthesis exhibited a maximum 8–12 h after elicitation in the cells of both cultivars. Concomitant with the fivefold-higher phytoalexin accumulation, CHS activity increased twofold in the cells of the resistant cultivar. The maximum of the elicitor-induced CHS-mRNA activity was determined 4 h after onset of induction in the cultures of both cultivars, although in cells of cultivar ILC 3279 this mRNA activity was induced at a level twofold higher than that in cells of the susceptible race ILC 1929. Investigations of CHS isoenzymes by two-dimensional gel electrophoresis of immunoprecipitated in-vitro-translated protein indicated the presence of five proteins. In the cells of both cultivars only two of the isoenzymes were induced after elicitor treatment. Analysis of the total in-vitro-translated proteins by two-dimensional gel electrophoresis showed that the constitutively expressed patterns of mRNA activities in the cell cultures of the two cultivars were identical. After elicitation, considerably more translatable mRNAs were induced in the cells of cultivar ILC 3279. The few induced proteins, and their respective mRNA activities, which could be detected in the cells of the susceptible cultivar, all existed in the cells of the resistant cultivar, too. One highly induced protein (M_r 18 kDa) found in the cells of cultivar ILC 3279 reached its maximum mRNA activity 6 h after elicitor application. The amount of this protein was hardly increased in the cells of the susceptible cultivar. This protein appears to be excreted from the cells into the growth medium.Abbreviations CHS chalcone synthase - IEF isoelectric focussing - ILC international legume chickpea - PR-protein pathogenesis-related protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. The authors thank Dr. K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for provision of antisera and the International Centre for Agricultural Research in the Dry Areas (Aleppo, Syria) for plant material.     

Intracellular location of NADP+-linked malic enzyme in C3 plants

Ultraviolet light induces anthocyanin biosynthesis in cell cultures of an Afghan cultivar of Daucus carota (Daucus carota L. ssp. sativus). Simultaneous treatment with a fungal elicitor from Pythium aphanidermatum results in an inhibition of the catalytic activity of chalcone synthase (CHS), which in turn correlates with an inhibition of anthocyanin biosynthesis. On immunoblots, one isoenzyme (40 kDa) of CHS disappears upon elicitor treatment. On an mRNA level, only the mRNA for the 40-kDa-CHS is active after treatment with ultraviolet light. After inhibition of anthocyanin biosynthesis by the elicitor the enzyme protein disappears and the CHS mRNA is strongly diminished. This inhibition depends on the concentration of the elicitor. In addition, elicitor treatment leads to an induction of the general phenylpropanoid pathway as well as to the accumulation of 4-hydroxybenzoic acid which is covalently bound to wall polysaccharides of the carrot cells. The possible function of phenylalanine ammonia-lyase in providing precursors for 4-hydroxybenzoic acid is discussed.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for providing us with antisera to CHS and PAL, respectively. This work was supported by a grant from the Deutsche Forschungsgemeinschaft and scholarships from the Friedrich-Ebert-Stiftung (J. G.), the Landesgraduierten-förderungsgesetz Baden-Württemberg (J.-P. S) and the Gerhard-Rösch-Stiftung (D. S.). We thank R. Hofmann for her excellent technical assistance.     

Changes in activities of enzymes involved in flavonoid metabolism during the initiation and suppression of anthocyanin synthesis in carrot suspension cultures regulated by 2,4-dichlorophenoxyacetic acid

When anthocyanin synthesis was induced in cell suspension cultures of carrot ( Daucus carota L. cv. Kurodagosun) by transfer to medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 6.-.-.-), and chalcone-flavanone isomerase (CHFI, EC 5.5.1.6) activities appeared, reaching maxima 6–7 days after transfer. The maximum specific activity of CHS was much lower than that of PAL or CHFI. In a medium containing 2,4-D, no anthocyanin was synthesized, PAL and CHFI activities were suppressed and CHS activity could not be detected at all. The activities of PAL and CHS in cells cultured without 2,4-D for 6 days began to decrease within 3–6 h of 2,4-D addition. CHS activity was completely repressed 24–36 h after the addition, but CHFI activity was almost unchanged at this time. After culture without 2,4-D for 6 days, cell suspensions were transferred to fresh media either lacking or containing 2,4-D. After transfer, PAL increased in both media within 3 h, whereas CHS activity and anthocyanin accumulation were coordinated and both were completely regulated by 2,4-D. Changes in CHS activity rather than PAL activity correlate with changes in anthocyanin accumulation under various culture conditions.     

Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Luc firefly luciferase - PAL phenylalanine ammonia-lyase - UV ultraviolet     

Stress responses in alfalfa (Medicago sativa L.). 8. Cis-elements and trans-acting factors for the quantitative expression of a bean chalcone synthase gene promoter in electroporated alfalfa protoplasts

A chimeric gene consisting of a bean (Phaseolus vulgaris L.) chalcone synthase (CHS) promoter fused to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed, and further induced by fungal elicitor, when electroporated into alfalfa (Medicago sativa L.) suspension cell protoplasts. Functional analysis of 5 deletions of the CHS promoter-CAT construct in these protoplasts indicated that the region between –326 and –130 contained both activator and silencer elements. Co-electroporation experiments confirmed that these cis-acting elements were binding sites for functionally active trans factors. In vitro DNase I footprinting revealed four potential binding sites for alfalfa suspension cell nuclear proteins between positions –326 and –130 of the CHS promoter. These sites mapped to regions shown to contain functional cis-acting elements on the basis of the deletion analysis. Three of these sites mapped to previously identified binding sites for bean nuclear proteins. Competition gel retardation analysis using oligonucleotide probes containing binding site sequences revealed sequence-specific binding of alfalfa nuclear proteins to an AT-rich element and a putative GT-1 factor consensus binding sequence. Our results define cis elements and their cognate trans factors functionally active in determining the quantitative expression of a defense response gene in a heterologous transient expression system.Abbreviations CAT chloramphenicol acetyltransferase - CHS chalcone synthase (EC 2.3.1.74) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Gene transfer into peanut (Arachis hypogaea L.) by Agrobacterium tumefaciens

Introduction of foreign genes into plant tissues via Agrobacterium tumefaciens based vectors requires specific knowledge of Agrobacterium-host compatibility. Therefore, to develop a transformation protocol for peanut (Arachis hypogaea L.), five Brazilian cultivars were screened with four wild-type A.tumefaciens strains. Successful transformation was dependent on specific bacterial strain-plant cultivar interactions and strain A281 was the most effective for tumor induction. Tumors displayed hormone autonomous growth, were opine positive and contained DNA that was homologous to the T-DNA of the inciting strain. Tumors induced on seed and seedling explants by A281 (pTD02) also expressed the reporter genes gus and npt-II contained in the binary vector. These results show that peanut is a permissive host for the acceptance of genes from specific A.tumefaciens gene vectors.Abbreviations GUS ß-glucuronidase (EC 3.2.1.31) - NPT-II neomycin phosphotransferase II (EC 2.7.1.95) - EDTA ethylene-diamine-tetracetic acid     

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.     

Sequence diversity and differential expression of major phenylpropanoid-flavonoid biosynthetic genes among three mango varieties

Background

Mango fruits contain a broad spectrum of phenolic compounds which impart potential health benefits; their biosynthesis is catalysed by enzymes in the phenylpropanoid-flavonoid (PF) pathway. The aim of this study was to reveal the variability in genes involved in the PF pathway in three different mango varieties Mangifera indica L., a member of the family Anacardiaceae: Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) and to determine associations with gene expression and mango flavonoid profiles.

Results

A close evolutionary relationship between mango genes and those from the woody species poplar of the Salicaceae family (Populus trichocarpa) and grape of the Vitaceae family (Vitis vinifera), was revealed through phylogenetic analysis of PF pathway genes. We discovered 145 SNPs in total within coding sequences with an average frequency of one SNP every 316 bp. Variety IW had the highest SNP frequency (one SNP every 258 bp) while KP and NDM had similar frequencies (one SNP every 369 bp and 360 bp, respectively). The position in the PF pathway appeared to influence the extent of genetic diversity of the encoded enzymes. The entry point enzymes phenylalanine lyase (PAL), cinnamate 4-mono-oxygenase (C4H) and chalcone synthase (CHS) had low levels of SNP diversity in their coding sequences, whereas anthocyanidin reductase (ANR) showed the highest SNP frequency followed by flavonoid 3’-hydroxylase (F3’H). Quantitative PCR revealed characteristic patterns of gene expression that differed between mango peel and flesh, and between varieties.

Conclusions

The combination of mango expressed sequence tags and availability of well-established reference PF biosynthetic genes from other plant species allowed the identification of coding sequences of genes that may lead to the formation of important flavonoid compounds in mango fruits and facilitated characterisation of single nucleotide polymorphisms between varieties. We discovered an association between the extent of sequence variation and position in the pathway for up-stream genes. The high expression of PAL, C4H and CHS genes in mango peel compared to flesh is associated with high amounts of total phenolic contents in peels, which suggest that these genes have an influence on total flavonoid levels in mango fruit peel and flesh. In addition, the particularly high expression levels of ANR in KP and NDM peels compared to IW peel and the significant accumulation of its product epicatechin gallate (ECG) in those extracts reflects the rate-limiting role of ANR on ECG biosynthesis in mango.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1784-x) contains supplementary material, which is available to authorized users.     

Dose responses for Colletotrichum lindemuthianum elicitor-mediated enzyme induction in French bean cell suspension cultures

The induction of L-phenylalanine ammonialyase (PAL, EC 4.3.1.5) and flavanone synthase in French bean cell suspension cultures in response to heat-released elicitor from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum is highly dependent upon elicitor concentration. The elicitor dose-response curve for PAL induction shows two maxima at around 17.5 and 50 g elicitor carbohydrate per ml culture, whereas the flavanone synthase response shows one maximum at around 100 g ml^-1. The PAL response is independent of the elicitor concentration present during the lag phase of enzyme induction; if the initial elicitor concentration is increased after 2 h by addition of extra elicitor, or decreased by dilution of the cultures, the dose response curves obtained reflect the concentration of elicitor present at the time of harvest. PAL induction is not prevented by addition of methyl sugar derivatives to the cultures; -methyl-D-glucoside, itself a weak elicitor of PAL activity, elicits a multiphasic PAL response when increasing concentrations are added in the presence of Colletotrichum elicitor. Eight fractions with different monosaccharide compositions, obtained from the crude elicitor by gel-filtration, each elicit different dose-responses for PAL induction; the response to unfractionated elicitor is not the sum of the response to the isolated fractions. There is no correlation between the ability of the fractions to induce PAL in the cultures and their ability to act as elicitors of isoflavonoid phytoalexin accumulation in bean hypocotyls.Abbreviations PAL phenylalanine ammonia-lyase - PMS Phytophthora megasperma var sojae