The repertoire of secondary metabolism (involving the production of compounds not essential for growth) in the plant kingdom is enormous, but the genetic and functional basis for this diversity is hard to analyse as many of the biosynthetic enzymes are unknown. We have now identified a key enzyme in the ornamental plant Gerbera hybrida (Asteraceae) that participates in the biosynthesis of compounds that contribute to insect and pathogen resistance. Plants transformed with an antisense construct of gchs2, a complementary DNA encoding a previously unknown function, completely lack the pyrone derivatives gerberin and parasorboside. The recombinant plant protein catalyses the principal reaction in the biosynthesis of these derivatives GCHS2 is a polyketide synthase that uses acetyl-CoA and two condensation reactions with malonyl-CoA to form the pyrone backbone of the natural products. The enzyme also accepts benzoly-CoA to synthesize the backbone of substances that have become of interest as inhibitors of the HIV-1 protease. GCHS2 is related to chalcone synthase (CHS) and its properties define a new class of function in the protein superfamily. It appears that CHS-related enzymes are involved in the biosynthesis of a much larger range of plant products than was previously realized
Chalcone (CHS) and stilbene (STS) synthases are related plant-specific polyketide synthases that are key enzymes in the biosynthesis of flavonoids and of stilbene phytoalexins, respectively. A phylogenetic tree constructed from 34 CHS and four STS sequences revealed that the STS formed no separate cluster but grouped with CHS from the same or related plants. This suggested that STS evolved from CHS several times independently. We attempted to stimulate this by site-directed mutagenesis of an interfamily CHS/STS hybrid, which contained 107 amino acids of a CHS from Sinapis alba (N-terminal) and 287 amino acids of a STS from Arachis hypogaea. The hybrid had no enzyme activity. Three amino acid exchanges in the CHS part (Gln-100 to Glu, Val-103 to Met, Val-105 to Arg) were sufficient to obtain low STS activity, and one additional exchange (Gly-23 to Thr) resulted in 20-25% of the parent STS activity. A kinetic analysis indicated (1) that the hybrids had the same Km for the substrate 4-coumaroyl-CoA but a lower Vmax than the parent STS, and (2) that they had a different substrate preference than the parent STS and CHS. Most of the other mutations and their combinations led to enzymatically inactive protein aggregates, suggesting that the subunit folding and/or the dimerization was disturbed. We propose that STS evolved from CHS by a limited number of amino acid exchanges, and that the advantage gained by this enzyme function favored the selection of plants with improved STS activity.
Gene 2 from the T region of Ti plasmids appears to be expressed both in eucaryotic and in procaryotic systems. In transformed plant cells it participates in auxin-controlled growth and differentiation, and in bacteria it is expressed into a defined protein of M, 49000. We investigated the possibility that it codes for an enzyme involved in auxin bi osynt hesis .Only extracts from Escherichia coli cells expressing gene 2 hydrolyzed indole-3-acetamide into a substance which was unambiguously identified as indole-3-acetic acid. The same reaction was found in Agrobacteria containing gene 2, but not in strains lacking the gene. Extracts from tobacco crown gall cells, but not from non-transformed cells, showed the same enzyme activity, and the reaction product was also identified as indole-3-acetic acid. The results indicate that gene 2 of the T region, which participates in tumorous growth of plant cells, codes both in bacteria and in plants for an amidohydrolase involved in the biosynthesis of the plant hormone indole-3-acetic acid.
A gene from groundnut (Arachis hypogaea) coding for stilbene synthase was transferred together with a chimaeric kanamycin resistance gene. It was found to be rapidly expressed after induction with UV light and elicitor in tobacco cells (Nicotiana tabacum). Comparative studies of stilbene synthase mRNA synthesis in groundnut and transgenic tobacco suspension cultures revealed the same kinetics of gene expression. Stilbene synthase specific mRNA was detectable 30 minutes after elicitor induction and 10 minutes after UV irradiation. The maximum of mRNA accumulation was between 2 and 8 hours post induction. 24 hours after induction stilbene synthase mRNA accumulation ceased. Furthermore, in transgenic tobacco plants, the gene was found to be inducible in sterile roots, stems and leaves. Stilbene synthase was demonstrated in crude protein extracts from transgenic tobacco cell cultures using specific antibodies. Resveratrol, the product of stilbene synthase, was identified by HPLC and antisera raised against resveratrol.
Resveratrol synthase (RS), a key enzyme in biosynthesis of stilbene-type phytoalexins, catalyzes the formation of resveratrol from coumaroyl-CoA and malonyl-CoA. Two cDNA clones, pGSCl and pGSC2, have been isolated from cDNA libraries established with poly(A)-rich RNA from peanut (Arachis hypogaea) cell cultures specifically induced for RS. These cDNAs were used to identify two genomic clones (pGSG10 and pGSG11). Sequence analysis shows that the two clones overlap in a large stretch of nearly identical sequences, and that pGSGlO contains the 5' and pGSGl1 the 3' end of RS genes. The sequences reveal a single intron, and the size of the predicted protein is 42.7 kDa, in close agreement with that observed in polyacrylamide gels (43 kDa). Chalcone synthase (CHS), a key enzyme of flavonoid biosynthesis, utilizes the same substrates as RS, but the product is different (naringenin chalcone). Comparison of RS with CHS consensus sequences shows that the two genes are related. Homology extends throughout the coding region, and the intron in RS is at the same position as a conserved intron in CHS. However, RS reveals a substantial number of amino acid differences to CHS in positions highly conserved in all CHS enzymes. It is proposed that the two proteins possess a common scaffold necessary for binding of the substrates and the type of enzyme reaction, and that the differences are responsible for the formation of different products.Stilbenes are constituents of a limited number of plants, and often their synthesis is induced by fungal attack or by other stress conditions. Several stilbenes possess phytoalexin properties [l -31, and their formation may be considered as part of the general defense mechanisms in such plants. Stilbene synthases are the key enzymes in stilbene synthesis, since they produce the backbone molecule which then may be modified by other enzymes. According to their substrate specificities, at least two different types can be distinguished. Both utilize malonyl-CoA as one substrate, but resveratrol synthase (RS, Fig. 1) prefers coumaroyl-CoA, while pinosylvin synthase prefers cinnamoyl-CoA as second substrate. The type of enzyme reaction is the same and the products differ only by the one hydroxyl group present in coumaroyl-CoA but absent in cinnamoyl-CoA [4]. RS from cell cultures of peanut (Arachis hypogaea) is the enzyme which has been studied in most detail [S]. The enzyme is inducible in these cultures [6, 71 and the cells produce the same group of stilbenes as peanut plants [S].Chalcone synthase (CHS), an enzyme important in flavonoid biosynthesis [9, lo], also utilizes coumaroyl-CoA and malonyl-CoA, and CHS and RS are likely to share basic mechanisms of catalysis. The products of the reactions, however, are different ( Fig. l ) and purified RS does not possess CHS activity [5]. The proteins are also immunologically different, since antisera against CHS do not react with RS, and vice versa [ll]. CHS clones from different plants have been described and comparisons indicate that DNA and amino
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