In livingstone daisy (Dorotheanthus bellidiformis ), betanidin 5‐O‐glucosyltransferase (UGT73A5) is involved in the regiospecific glucosylation of betanidin and various flavonols. Based on sequence alignments several amino acid candidates which might be essential for catalysis were identified. The selected amino acids of the functionally expressed protein, suggested to be involved in substrate binding and turnover, were substituted via site‐directed mutagenesis. The substitution of two highly conserved amino acids, Glu378, located in the proposed UDP‐glucose binding site, and His22, located close to the N‐terminus, led to the complete loss of enzyme activity. A 3D model of this regiospecific betanidin and flavonoid glucosyltransferase was constructed and the active site modelled. This model was based on the crystallographic structure of a bacterial UDP‐glucose‐dependent glucosyltransferase from Amycolatopsis orientalis used as a template and the generated null mutations. To explain the observed inversion in the configuration of the bound sugar, semiempirical calculations favour an SN‐1 reaction, as one plausible alternative to the generally proposed SN‐2 mechanism discussed for plant natural product glucosyltransferases. The calculated structural data do not only explain the abstraction of a proton from the acceptor betanidin, but further imply that the reaction mechanism might also involve a catalytic triad, with similarities described for the serine protease family.

The fatty acid hydroperoxide (HP) substrates required for the biosynthesis of jasmonic acid (JA) and green leaf volatiles (GLVs) are supplied by separate lipoxygenases (LOX). We silenced the expression of two genes downstream of the LOX: allene oxide synthase (AOS) and HP lyase (HPL) by antisense expression of endogenous genes (NaAOS , NaHPL ) in Nicotiana attenuata , in which the biosynthesis of JA is amplified by herbivore‐specific elicitors. We report that these elicitors also amplify wound‐induced GLV releases, but suppress the wound‐induced increase of NaHPL transcripts, suggesting that substrate flux controls GLV biosynthesis. As expected, silencing of NaHPL and NaAOS reduced GLV release and JA accumulation, respectively. Surprisingly, HPL‐ and AOS‐silenced plants had enhanced JA and GLV responses, suggesting substrate ‘crosstalk’ between these two oxylipin cascades. Plants with depleted GLVs (as‐hpl ) were less attractive than wild type (WT) or empty vector control plants in choice‐tests with native lepidopteran herbivores. In feeding trials, Manduca sexta larvae developed slower on as‐hpl plants. The reduced larval consumption and performance, which was not caused by increases in defense responses in as‐hpl plants, could be restored to WT levels by the addition of synthetic GLVs, demonstrating that GLVs function as feeding stimulants. Gene expression profiling by cDNA microarray analysis and characterization of several induced defenses in herbivore‐elicited as‐hpl and as‐aos plants revealed differential involvement of JA and GLVs in defense signaling. Elicitation of volatile terpenoids (an indirect defense) requires JA signaling, where as trypsin protease inhibitor elicitation (a direct defense) requires both functional JA and GLV cascades.

Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. Here, we characterize a putative UDP‐glucose:thiohydroximate S‐glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochemical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the thiohydroximate functional group. Low K m values for phenylacetothiohydroximic acid (approximately 6 μ m ) and UDP‐glucose (approximately 50 μm ) strongly suggest that thiohydroximates are in vivo substrates of UGT74B1. Insertional loss‐of‐function ugt74b1 mutants exhibit significantly decreased, but not abolished, glucosinolate accumulation. In addition, ugt74b1 mutants display phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in light‐grown plants, excess adventitious rooting and incomplete leaf vascularization. Indeed, during early plant development, mutant ugt74b1 seedlings accumulate nearly threefold more indole‐3‐acetic acid than the wild type. Other phenotypes, however, such as chlorosis along the leaf veins, are likely caused by thiohydroximate toxicity. Analysis of UGT74B1 promoter activity during plant development reveals expression patterns consistent with glucosinolate metabolism and induction by auxin treatment. The results are discussed in the context of known mutations in glucosinolate pathway genes and their effects on auxin homeostasis. Taken together, our work provides complementary in vitro and in vivo evidence for a primary role of UGT74B1 in glucosinolate biosynthesis.

Members of the Brassicaceae family accumulate specific sinapate esters, i.e. sinapoylcholine (sinapine), which is considered as a major antinutritive compound in seeds of important crop plants like Brassica napus , and sinapoylmalate, which is implicated in UV‐B tolerance in leaves. We have studied the molecular regulation of the sinapate ester metabolism in B. napus , and we describe expression of genes, some properties of the encoded proteins and profiles of the metabolites and enzyme activities. The cloned cDNAs encoding the key enzymes of sinapine biosynthesis, UDP‐glucose (UDP‐Glc):B. napus sinapate glucosyltransferase (BnSGT1) and sinapoylglucose:B. napus choline sinapoyltransferase (BnSCT), were functionally expressed. BnSGT1 belongs to a subgroup of plant GTs catalysing the formation of 1‐O‐hydroxycinnamoyl‐β‐d ‐glucoses. BnSCT is another member of serine carboxypeptidase‐like (SCPL) family of acyltransferases. The B. napus genome contains at least two SGT and SCT genes, each derived from its progenitors B. oleracea and B. rapa . BnSGT1 and BnSCT activities are subjected to pronounced transcriptional regulation. BnSGT1 transcript level increases throughout early stages of seed development until the early cotyledonary stage, and stays constant in later stages. The highest level of BnSGT1 transcripts is reached in 2‐day‐old seedlings followed by a dramatic decrease. In contrast, expression of BnSCT is restricted to developing seeds. Regulation of gene expression at the transcript level seems to be responsible for changes of BnSGT1 and BnSCT activities during seed and seedling development of B. napus . Together with sinapine esterase (SCE) and sinapoylglucose:malate sinapoyltransferase (SMT), activities of BnSGT1 and BnSCT show a close correlation with the accumulation kinetics of the corresponding metabolites.

The hyperaccumulation of zinc (Zn) and cadmium (Cd) is a constitutive property of the metallophyte Arabidopsis halleri . We therefore used Arabidopsis GeneChips to identify genes more active in roots of A. halleri as compared to A. thaliana under control conditions. The two genes showing highest expression in A. halleri roots relative to A. thaliana roots out of more than 8000 genes present on the chip encode a nicotianamine (NA) synthase and a putative Zn2+ uptake system. The significantly higher activity of these and other genes involved in metal homeostasis under various growth conditions was confirmed by Northern and RT‐PCR analyses. A. halleri roots also show higher NA synthase protein levels. Furthermore, we developed a capillary liquid chromatography electrospray ionization quadrupole time‐of‐flight mass spectrometry (CapLC‐ESI‐QTOF‐MS)‐based NA analysis procedure and consistently found higher NA levels in roots of A. halleri . Expression of a NA synthase in Zn2+‐hypersensitive Schizosaccharomyces pombe cells demonstrated that formation of NA can confer Zn2+ tolerance. Taken together, these observations implicate NA in plant Zn homeostasis and NA synthase in the hyperaccumulation of Zn by A. halleri . Furthermore, the results show that comparative microarray analysis of closely related species can be a valuable tool for the elucidation of phenotypic differences between such species.

Plants have evolved complex strategies to maintain phosphate (Pi) homeostasis and to maximize Pi acquisition when the macronutrient is limiting. Adjustment of root system architecture via changes in meristem initiation and activity is integral to the acclimation process. However, the mechanisms that monitor external Pi status and interpret the nutritional signal remain to be elucidated. Here, we present evidence that the Pi deficiency response , pdr2 , mutation disrupts local Pi sensing. The sensitivity and amplitude of metabolic Pi‐starvation responses, such as Pi‐responsive gene expression or accumulation of anthocyanins and starch, are enhanced in pdr2 seedlings. However, the most conspicuous alteration of pdr2 is a conditional short‐root phenotype that is specific for Pi deficiency and caused by selective inhibition of root cell division followed by cell death below a threshold concentration of about 0.1 mm external Pi. Measurements of general Pi uptake and of total phosphorus (P) in root tips exclude a defect in high‐affinity Pi acquisition. Rescue of root meristem activity in Pi‐starved pdr2 by phosphite (Phi), a non‐metabolizable Pi analog, and divided‐root experiments suggest that pdr2 disrupts sensing of low external Pi availability. Thus, PDR2 is proposed to function at a Pi‐sensitive checkpoint in root development, which monitors environmental Pi status, maintains and fine‐tunes meristematic activity, and finally adjusts root system architecture to maximize Pi acquisition.

Changing environmental conditions, atmospheric pollutants and resistance reactions to pathogens cause production of reactive oxygen species (ROS) in plants. ROS in turn trigger the activation of signaling cascades such as the mitogen‐activated protein kinase (MAPK) cascade and accumulation of plant hormones, jasmonic acid, salicylic acid (SA), and ethylene (ET). We have used ozone (O3) to generate ROS in the apoplast of wild‐type Col‐0 and hormonal signaling mutants of Arabidopsis thaliana and show that this treatment caused a transient activation of 43 and 45 kDa MAPKs. These were identified as AtMPK3 and AtMPK6. We also demonstrate that initial AtMPK3 and AtMPK6 activation in response to O3 was not dependent on ET signaling, but that ET is likely to have secondary effects on AtMPK3 and AtMPK6 function, whereas functional SA signaling was needed for full‐level AtMPK3 activation by O3. In addition, we show that AtMPK3 , but not AtMPK6 , responded to O3 transcriptionally and translationally during O3 exposure. Finally, we show in planta that activated AtMPK3 and AtMPK6 are translocated to the nucleus during the early stages of O3 treatment. The use of O3 to induce apoplastic ROS formation offers a non‐invasive in planta system amenable to reverse genetics that can be used for the study of stress‐responsive MAPK signaling in plants.