The benzoxazinoid biosynthetic pathway is of monophyletic origin in the Poaceae. In the dicots
Aphelandra squarrosa,
Consolida orientalis, and
Lamium galeobdolon, benzoxazinoid biosynthesis is ...analogously organised: The branchpoint is established by a homolog of the alpha-subunit of tryptophan synthase, P450 enzymes catalyse hydroxylations and at least the first hydroxylation reaction is identical in dicots and Poaceae. The toxic aglucon is glucosylated by an UDP-glucosyltransferase.
Benzoxazinoids are secondary metabolites that are effective in defence and allelopathy. They are synthesised in two subfamilies of the Poaceae and sporadically found in single species of the dicots. The biosynthesis is fully elucidated in maize; here the genes encoding the enzymes of the pathway are in physical proximity. This “biosynthetic cluster” might facilitate coordinated gene regulation. Data from
Zea mays,
Triticum aestivum and
Hordeum lechleri suggest that the pathway is of monophyletic origin in the Poaceae. The branchpoint from the primary metabolism (
Bx1 gene) can be traced back to duplication and functionalisation of the alpha-subunit of tryptophan synthase (TSA). Modification of the intermediates by consecutive hydroxylation is catalysed by members of a cytochrome P450 enzyme subfamily (
Bx2–
Bx5). Glucosylation by an UDP-glucosyltransferase (UGT,
Bx8,
Bx9) is essential for the reduction of autotoxicity of the benzoxazinoids. In some species 2,4-dihydroxy-1,4-benzoxazin-3-one-glucoside (DIBOA-glc) is further modified by the 2-oxoglutarate-dependent dioxygenase BX6 and the
O-methyltransferase BX7. In the dicots
Aphelandra squarrosa,
Consolida orientalis, and
Lamium galeobdolon, benzoxazinoid biosynthesis is analogously organised: The branchpoint is established by a homolog of TSA, P450 enzymes catalyse hydroxylations and at least the first hydroxylation reaction is identical in dicots and Poaceae, the toxic aglucon is glucosylated by an UGT. Functionally, TSA and BX1 are indole-glycerolphosphate lyases (IGLs).
Igl genes seem to be generally duplicated in angiosperms. Modelling and biochemical characterisation of IGLs reveal that the catalytic properties of the enzyme can easily be modified by mutation. Independent evolution can be assumed for the BX1 function in dicots and Poaceae.
When attacked by herbivorous insects, plants emit volatile compounds that attract natural enemies of the insects. It has been proposed that these volatile signals can be manipulated to improve crop ...protection. Here, we demonstrate the full potential of this strategy by restoring the emission of a specific belowground signal emitted by insect-damaged maize roots. The western corn rootworm induces the roots of many maize varieties to emit (E)-β-caryophyllene, which attracts entomopathogenic nematodes that infect and kill the voracious root pest. However, most North American maize varieties have lost the ability to emit (E)-β-caryophyllene and may therefore receive little protection from the nematodes. To restore the signal, a nonemitting maize line was transformed with a (E)-β-caryophyllene synthase gene from oregano, resulting in constitutive emissions of this sesquiterpene. In rootworm-infested field plots in which nematodes were released, the (E)-β-caryophyllene-emitting plants suffered significantly less root damage and had 60% fewer adult beetles emerge than untransformed, nonemitting lines. This demonstration that plant volatile emissions can be manipulated to enhance the effectiveness of biological control agents opens the way for novel and ecologically sound strategies to fight a variety of insect pests.
Polyploidy, the presence of more than two complete sets of chromosomes in an organism, has significantly shaped the genomes of angiosperms during evolution. Two forms of polyploidy are often ...considered: allopolyploidy, which originates from interspecies hybrids, and autopolyploidy, which originates from intraspecies genome duplication events. Besides affecting genome organization, polyploidy generates other genetic effects. Synthetic allopolyploid plants exhibit considerable transcriptome alterations, part of which are likely caused by the reunion of previously diverged regulatory hierarchies. In contrast, autopolyploids have relatively uniform genomes, suggesting lower alteration of gene expression. To evaluate the impact of intraspecies genome duplication on the transcriptome, we generated a series of unique Arabidopsis thaliana autotetraploids by using different ecotypes. A. thaliana autotetraploids show transcriptome alterations that strongly depend on their parental genome composition and include changed expression of both new genes and gene groups previously described from allopolyploid Arabidopsis. Alterations in gene expression are stable, nonstochastic, developmentally specific, and associated with changes in DNA methylation. We propose that Arabidopsis possesses an inherent and heritable ability to sense and respond to elevated, yet balanced chromosome numbers. The impact of natural variation on alteration of autotetraploid gene expression stresses its potential importance in the evolution and breeding of plants.
Benzoxazinoids represent preformed protective and allelophatic compounds that are found in a multitude of species of the family Poaceae (Gramineae) and occur sporadically in single species of ...phylogenetically unrelated dicots. Stabilization by glucosylation and activation by hydrolysis is essential for the function of these plant defense compounds. We isolated and functionally characterized from the dicot larkspur (Consolida orientalis) the benzoxazinoid-specific UDP-glucosyltransferase and ß-glucosidase that catalyze the enzymatic functions required to avoid autotoxicity and allow activation upon challenge by herbivore and pathogen attack. A phylogenetic comparison of these enzymes with their counterparts in the grasses indicates convergent evolution by repeated recruitment from homologous but not orthologous genes. The data reveal a great evolutionary flexibility in recruitment of these essential functions of secondary plant metabolism.
Heterosis, the greater vigor of hybrids compared to their parents, has been exploited in maize breeding for more than 100 years to produce ever better performing elite hybrids of increased yield. ...Despite extensive research, the underlying mechanisms shaping the extent of heterosis are not well understood, rendering the process of selecting an optimal set of parental lines tedious. This study is based on a dataset consisting of 112 metabolite levels in young roots of four parental maize inbred lines and their corresponding twelve hybrids, along with the roots' biomass as a heterotic trait. Because the parental biomass is a poor predictor for hybrid biomass, we established a model framework to deduce the biomass of the hybrid from metabolite profiles of its parental lines. In the proposed framework, the hybrid metabolite levels are expressed relative to the parental levels by incorporating the standard concept of additivity/dominance, which we name the Combined Relative Level (CRL). Our modeling strategy includes a feature selection step on the parental levels which are demonstrated to be predictive of CRL across many hybrid metabolites. We demonstrate that these selected parental metabolites are further predictive of hybrid biomass. Our approach directly employs the diallel structure in a multivariate fashion, whereby we attempt to not only predict macroscopic phenotype (biomass), but also molecular phenotype (metabolite profiles). Therefore, our study provides the first steps for further investigations of the genetic determinants to metabolism and, ultimately, growth. Finally, our success on the small-scale experiments implies a valid strategy for large-scale experiments, where parental metabolite profiles may be used together with profiles of selected hybrids as a training set to predict biomass of all possible hybrids.
Two classes of tryptophan synthase beta genes have been retained since the origin of plants. Both types of enzymes are functionally expressed in Arabidopsis and maize.
Tryptophan synthase β-subunits ...(TSBs) catalyze the last step in tryptophan biosynthesis, i.e. the condensation of indole and serine yielding tryptophan. In microorganisms two subfamilies of TSBs (here designated as type 1 and type 2) are known, which are only distantly related. Surprisingly, in all genomes of multicellular plants analyzed genes encoding both types are present. While type 1 enzymes are well established as components of tryptophan synthase complexes, type 2 enzymes in plants have not yet been characterized. Tissue specific expression of the
TSB genes from
Arabidopsis thaliana was analyzed. While
AtTSB1 is the predominantly expressed isoform in vegetative tissues,
AtTSB1 and
AtTSBtype2 reach similar transcript levels in seeds. AtTSBtype2 protein was expressed in
Escherichia coli and purified. It converted indole and serine to tryptophan with a strikingly low
K
m-value for indole of ca. 74
nM.
Attsbtype2 T-DNA insertion mutants showed no obvious deviation from the wild type phenotype, indicating that AtTSBtype2 function is not essential under standard growth conditions. As example for a monocot enzyme, maize
TSB
type 2 was analyzed and found to be transcribed in various tissues. ZmTSBtype2 was also catalytically active and here a
K
m-value for indole of ca. 7
μM was determined. These data indicate that TSB type 2 enzymes generally are functionally expressed in plants. Their potential biological role is discussed.
The synthesis of a diverse spectrum of secondary metabolites has allowed plants to develop sophisticated chemical defense mechanisms. Maize (Zea mays L.), for example, releases a cocktail of volatile ...compounds when attacked by a caterpillar. These compounds attract a parasitic wasp, which deposits its eggs in the larvae, thereby controlling the population size of the herbivore. Indole, which is part of the cocktail, is produced by an enzyme recruited from primary metabolism. Indole can either function as a volatile signal or be converted by specific cytochrome P450 enzymes into benzoxazinoids, which function as important defense chemicals.
Summary
Benzoxazinoids are major compounds involved in chemical defence in grasses. These toxins are stored in the vacuole as glucosides. Two glucosyltransferases, BX8 and BX9, that catalyse this ...last step of benzoxazinoid biosynthesis have been isolated via functional cloning. No close relative of these maize genes was found among the known glucosyltransferases. The enzymes display a very high degree of substrate specificity. DIMBOA, the major benzoxazinoid in young maize, is the preferred substrate. Both genes are highly expressed in young maize seedlings, the developmental stage with the highest activity of benzoxazinoid biosynthesis. Bx8 is included in the cluster of DIMBOA biosynthesis genes located on the short arm of chromosome 4. Hence, the gene cluster comprises three different enzymatic functions and a complete set of genes for the biosynthesis of DIBOA glucoside. Bx9 mapped to chromosome 1. Expression of Bx8 and Bx9 in Arabidopsis corroborated the potency of the enzymes in detoxification of their substrates. This capacity might have implications for allelopathic interactions.
Two indole alkaloids with defense related functions are synthesized in the genus
Hordeum of the Triticeae, gramine (3(dimethyl-amino-methyl)-indole) and the benzoxazinoid 2,4-dihydroxy-2
...H-1,4-benzoxazin-3(4
H)-one (DIBOA).
Two indole alkaloids with defense related functions are synthesized in the genus
Hordeum of the Triticeae. Gramine (3(dimethyl-amino-methyl)-indole) is found in
H. spontaneum and in some varieties of
H. vulgare, the benzoxazinoid 2,4-dihydroxy-2
H-1,4-benzoxazin-3(4
H)-one (DIBOA) is detected in
H. roshevitzii,
H. brachyantherum,
H. flexuosum,
H. lechleri. Biosynthesis of DIBOA and of gramine was found to be mutually exclusive in wild
Hordeum species, indicating that there was selection against simultaneous expression of both pathways during evolution. The full set of genes required for DIBOA biosynthesis in
H.lechleri was isolated and the respective enzyme functions were analyzed by heterologous expression. The cytochrome P450 genes
Bx2–
Bx5 demonstrate a monophyletic origin for
H. lechleri,
Triticum aestivum and
Zea mays.
HlBx2–
HlBx5 share highest homology to the orthologous genes of
T. aestivum. In contrast, the branch point enzyme of the DIBOA pathway, the indole-3-glycerol phosphate lyase BX1, might have evolved independently in
H. lechleri. In all
Hordeum species that synthesize DIBOA, DNA sequences homologous to
Bx genes are found. In contrast, these sequences are not detectable in the genomes of
H. vulgare and
H. spontaneum that do not synthesize benzoxazinoids.
Arabidopsis thaliana has, in conjunction with A. arenosa, developed into a system for the molecular analysis of alloplolyploidy. However, there are very few Arabidopsis lines available to study ...autopolyploidy. In order to investigate polyploidy on a reliable basis, we have optimised conventional methodologies and developed a novel strategy for the rapid generation and identification of polyploids based on trichome branching patterns. The analysis of more than two dozen independently induced Arabidopsis lines has led to interesting observations concerning the relationship between cell size and ploidy levels and on the relative stability of tetraploidy in Arabidopsis over at least three consecutive generations. The most important finding of this work is that neo-tetraploid lines exhibit considerable stability through all the generations tested. The systematic generation of tetraploid collections through this strategy as well as the lines generated in this work will help to unravel the consequences of polyploidy, particularly tetraploidy, on the genome, on gene expression and on natural diversity in Arabidopsis.
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