Summary

1. Induced resistance of plants to incompatible races of their microbial pathogens is often characterized by rapid cell death (the hypersensitive response) and the accumulation of low-Mr antimicrobial compounds termed phytoalexins. There is much indirect evidence to support a major role for phytoalexin induction as an event determining host resistance, and genetical analysis of race-specific pathogen-host interactions predicts that induced resistance occurs via interaction between a pathogen avirulence gene product and a host resistance gene product. Elicitors (inducing agents) of phytoalexin accumulation are possible candidates for the active products directly or indirectly resulting from the expression of microbial avirulence genes. The present review considers the process of phytoalexin induction, at the molecular level, from the fungal elicitor to the early changes in host gene expression associated with its action.



2. A number of microbial polysaccharides, glycoproteins, pectic enzymes, peptides and fatty acids have potent elicitor activity.



3. The results of studies on the purification and characterization of microbial elicitors depend on the nature of the bioassay used. Methods for elicitor extraction may lead to artifacts or loss of race specificity.



4. Elicitors isolated from plant pathogens may be race-specific or race-non-specific. In cases where only race-non-specific elicitors can be shown, race-specific induction of phytoalexins may result from the action of enhancer or suppressor molecules. Fungal glucans have been proposed as candidates to act in both these roles.



5. Molecular genetic approaches to the identification of phytopathogenic bacterial avirulence genes may help to prove or disprove the role of elicitors of the phytoalexin response as agents responsible for the induction of host resistance. Similar analyses, involving genetic transformation, should soon be possible for phytopathogenic fungi.



6. Studies on the nature of host receptors for microbial elicitors are still in their infancy. Such receptors are probably localized in the plant plasma membrane, and elicitation results in often striking changes in host membrane properties.



7. Cyclic 3′,5′-adenosine monophosphate and polyamines do not appear to act in plants as intracellular transducers of the phytoalexin response. Interest is now being shown in a possible role for calcium in intracellular signalling.



8. Plant cells contain endogenous elicitor molecules whose synthesis or release may play a role in the intercellular transmission of the phytoalexin response. The main candidates for endogenous elicitors are pectic fragments of the host cell wall, although no direct evidence for their involvement in plant-pathogen interactions is available. Pectic fragments may act as synergists in parallel with, rather than as couplers in series with, microbial elicitors. The role of ethylene as a response coupler for induced resistance phenomena is difficult to assess.



9. Some elicitors may themselves move to the host cell nucleus, although whether this is their effective site of action remains unclear.



10. Induction of the phytoalexin response is associated with specific changes in host gene expression related to the selective induction of new mRNA species and enzyme activities.



11. Progress is now being made in the characterization of enzymes specific for phytoalexin biosynthesis. Enzyme induction and/or infection appears to involve rapid modulation of gene transcription, although some post-translational events may also be involved in determining induction patterns.



12. Work is now commencing on the sequencing of the genes encoding elicitor-inducible enzymes. Multigene families have been identified for phenylalanine ammonia-lyase and chalcone synthase.



13. Phytoalexin accumulation may be accompanied by the rapid induction of ethylene biosynthesis and the expression of genes encoding activities involved in the synthesis of hydroxyproline-rich glycoproteins.



14. Pectic endogenous elicitors of phytoalexin accumulation are also active as inducers of systemic proteinase inhibitor synthesis in some solanaceous species.



15. Results of studies on the mechanisms of signal transmission and differential gene expression in relation to active defence reactions should be of relevance to many other aspects of the plant's response to environmental stimuli at the physiological, biochemical and molecular genetical levels.

The phytoalexin response: elicitation, signalling and control of host gene expression

Flavonols are important ultraviolet light protectants in many plants and contribute substantially to the quality and health-promoting effects of fruits and derived plant products. To study the regulation of flavonol synthesis in fruit, we isolated and characterized the grapevine (Vitis vinifera 'Shiraz') R2R3-MYB transcription factor VvMYBF1. Transient reporter assays established VvMYBF1 to be a specific activator of flavonol synthase1 (VvFLS1) and several other promoters of grapevine and Arabidopsis (Arabidopsis thaliana) genes involved in flavonol synthesis. Expression of VvMYBF1 in the Arabidopsis mutant myb12 resulted in complementation of its flavonol-deficient phenotype and confirmed the function of VvMYBF1 as a transcriptional regulator of flavonol synthesis. Transcript analysis of VvMYBF1 throughout grape berry development revealed its expression during flowering and in skins of ripening berries, which correlates with the accumulation of flavonols and expression of VvFLS1. In addition to its developmental regulation, VvMYBF1 expression was light inducible, implicating VvMYBF1 in the control of VvFLS1 transcription. Sequence analysis of VvMYBF1 and VvFLS1 indicated conserved putative light regulatory units in promoters of both genes from different cultivars. By analysis of the VvMYBF1 amino acid sequence, we identified the previously described SG7 domain and an additional sequence motif conserved in several plant MYB factors. The described motifs have been used to identify MYB transcription factors from other plant species putatively involved in the regulation of flavonol biosynthesis. To our knowledge, this is the first functional characterization of a light-inducible MYB transcription factor controlling flavonol synthesis in fruit.

The Grapevine R2R3-MYB Transcription Factor VvMYBF1 Regulates Flavonol Synthesis in Developing Grape Berries

A rapid and substantial progress to a broad understanding of flavonoid biosynthesis marked the previous reviews of this series of books (Hahlbrock and Grisebach, 1975; Ebel and Hahlbrock, 1982; Heller and Forkmann, 1988), and several other reviews have also appeared (Harborne, 1988; Zaprometov, 1989; Stafford, 1990). Advances achieved in the past few years are restricted to specific steps, filling some of the remaining gaps in this metabolic pathway. Among these reactions are the formation of 3-deoxyanthocyanins and 5-deoxyflavonoids, and the synthesis of the structurally derived pterocarpans, which represent the most prominent group of flavonoid phytoalexins. Among the flavonoid modifications, enzymatic formation of flavonoid sulfate esters and aromatic acylation of anthocyanins with hydroxycinnamic acid glucose esters as donors can be mentioned. Yet the key reaction to one of the main flavonoid classes, the anthocyanins, as well as the formation of epicatechin, the proanthocyanidins and some minor flavonoid-related compounds, such as aurones and dihydrochalcones, still remains a matter of debate.

Biosynthesis of flavonoids

Plant systems utilize a diverse array of cytochrome P450 monooxygenases (P450s) in their biosynthetic and detoxification pathways. The classic forms of these enzymes are heme-dependent mixed function oxidases that utilize NADPH or NADH and molecular oxygen to produce functionalized organic products. The nonclassical forms are monooxygenases that either do not utilize flavoproteins for dioxygen activation or fail to incorporate molecular oxygen into their final product. Biosynthetic P450s play paramount roles in the synthesis of lignin intermediates, sterols, terpenes, flavonoids, isoflavonoids, furanocoumarins, and a variety of other secondary plant products. Other catabolic P450s metabolize toxic herbicides and insecticides into nontoxic products or, conversely, activate nontoxic substances into toxic products. Biochemical and molecular characterizations on a number of plant P450s have indicated that the relationships between these heme proteins and their substrates are at least as complex as th...

Plant Cytochrome P450 Monooxygenases

Plant cytochrome P450.

A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen-dependent hydroxylation of (S)-naringenin [(S)-5, 7, 4'-trihydroxyflavanone] to eriodictyol (5, 7, 3', 4'-tetrahydroxyflavanone). Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3'-hydroxylase reaction. In contrast prunin (naringenin 7-O-beta-glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4-hydroxylase, did not catalyse hydroxylation of 4-coumaric acid to caffeic acid. 3'-Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P-450-dependent flavonoid 3'-monooxygenase. Pronounced differences in the inhibition of flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase were found with EDTA, potassium cyanide and N-ethylmaleimide. Irradiation of the cell cultures led to increase of flavonoid 3'-hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease. This is similar to light-induction of phenylalanine ammonialyase and cinnamate 4-hydroxylase. In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3'-hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and NADPH--cytochrome reductase. The results prove that flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase are two different microsomal monooxygenases.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07601.x

Induction and Characterization of a Microsomal Flavonoid 3′-Hydroxylase from Parsley Cell Cultures

A microsomal preparation from elicitor-challenged soybean cell suspension cultures catalyzes an NADPH-dependent and dioxygen-dependent 6a-hydroxylation of 3,9-dihydroxypterocarpan to 3,6a,9-trihydroxypterocarpan. The latter is a precursor for the soybean phytoalexin glyceollin. No reaction is observed with NADH. The 6a-hydroxylase is inhibited by cytochrome c.



Optical rotatory dispersion spectra of the enzymatic product formed from racemic dihydroxypterocarpan and of the remaining unreacted substrate proved that the product has the natural (6aS, 11aS)-configuration and that hydroxylation proceeds with retention of configuration.



The 6a-hydroxylase was also found in elicitor-challenged soybean seedlings. The results indicate that the 6a-hydroxylase is specifically involved in the biosynthesis of glyceollin.

Marie-Luise Hagmann

Papers

Induction and Characterization of a Microsomal Flavonoid 3′-Hydroxylase from Parsley Cell Cultures

Induction of phytoalexin synthesis in soybean. Stereospecific 3,9‐dihydroxypterocarpan 6a‐hydroxylase from elicitor‐induced soybean cell cultures