The seeds of most members of the Brassicaceae accumulate high amounts of sinapine (sinapoylcholine) that is rapidly hydrolyzed during early stages of seed germination. One of three isoforms of sinapine esterase activity (BnSCE3) has been isolated from Brassica napus seedlings and subjected to trypsin digestion and spectrometric sequencing. The peptide sequences were used to isolate BnSCE3 cDNA, which was shown to contain an open reading frame of 1170 bp encoding a protein of 389 amino acids, including a leader peptide of 25 amino acids. Sequence comparison identified the protein as the recently cloned BnLIP2, i.e. a GDSL lipase‐like protein, which displays high sequence identity to a large number of corresponding plant proteins, including four related Arabidopsis lipases. The enzymes belong to the SGNH protein family, which use a catalytic triad of Ser‐Asp‐His, with serine as the nucleophile of the GDSL motif. The corresponding B. napus and Arabidopsis genes were heterologously expressed in Nicotiana benthamiana leaves and proved to confer sinapine esterase activity. In addition to sinapine esterase activity, the native B. napus protein (BnSCE3/BnLIP2) showed broad substrate specificity towards various other choline esters, including phosphatidylcholine. This exceptionally broad substrate specificity, which is common to a large number of other GDSL lipases in plants, hampers their functional analysis. However, the data presented here indicate a role for the GDSL lipase‐like BnSCE3/BnLIP2 as a sinapine esterase in members of the Brassicaceae, catalyzing hydrolysis of sinapine during seed germination, leading, via 1‐O ‐sinapoyl‐β‐glucose, to sinapoyl‐l ‐malate in the seedlings.

Tropane alkaloids typically occur in the Solanaceae and are also found in Cochlearia officinalis , a member of the Brassicaceae. Tropinone reductases are key enzymes of tropane alkaloid metabolism. Two different tropinone reductases form one stereoisomeric product each, either tropine for esterified alkaloids or pseudotropine that is converted to calystegines. A cDNA sequence with similarity to known tropinone reductases (TR) was cloned from C. officinalis . The protein was expressed in Escherichia coli , and found to catalyze the reduction of tropinone. The enzyme is a member of the short‐chain dehydrogenase enzyme family and shows broad substrate specificity. Several synthetic ketones were accepted as substrates, with higher affinity and faster enzymatic turnover than observed for tropinone. C. officinalis TR produced both the isomeric alcohols tropine and pseudotropine from tropinone using NADPH + H+ as co‐substrate. Tropinone reductases of the Solanaceae, in contrast, are strictly stereospecific and form one tropane alcohol only. The Arabidopsis thaliana homologue of C. officinalis TR showed high sequence similarity, but did not reduce tropinone. A tyrosine residue was identified in the active site of C. officinalis TR that appeared responsible for binding and orientation of tropinone. Mutagenesis of the tyrosine residue yielded an active reductase, but with complete loss of TR activity. Thus C. officinalis TR presents an example of an enzyme with relaxed substrate specificity, like short‐chain dehydrogenases, that provides favorable preconditions for the evolution of novel functions in biosynthetic sequences.

The first step of the plastidial methylerythritol phosphate (MEP) pathway is catalyzed by two isoforms of 1‐deoxy‐d‐ xylulose 5‐phosphate synthase (DXS1 and DXS2). In Medicago truncatula , MtDXS1 and MtDXS2 genes exhibit completely different expression patterns. Most prominently, colonization by arbuscular mycorrhizal (AM) fungi induces the accumulation of certain apocarotenoids (cyclohexenone and mycorradicin derivatives) correlated with the expression of MtDXS2 but not of MtDXS1. To prove a distinct function of DXS2, a selective RNAi approach on MtDXS2 expression was performed in transgenic hairy roots of M. truncatula. Repression of MtDXS2 consistently led to reduced transcript levels in mycorrhizal roots, and to a concomitant reduction of AM‐induced apocarotenoid accumulation. The transcript levels of MtDXS1 remained unaltered in RNAi plants, and no phenotypical changes in non‐AM plants were observed. Late stages of the AM symbiosis were adversely affected, but only upon strong repression with residual MtDXS2‐1 transcript levels remaining below approximately 10%. This condition resulted in a strong decrease in the transcript levels of MtPT4 , an AM‐specific plant phosphate transporter gene, and in a multitude of other AM‐induced plant marker genes, as shown by transcriptome analysis. This was accompanied by an increased proportion of degenerating and dead arbuscules at the expense of mature ones. The data reveal a requirement for DXS2‐dependent MEP pathway‐based isoprenoid products to sustain mycorrhizal functionality at later stages of the symbiosis. They further validate the concept of a distinct role for DXS2 in secondary metabolism, and offer a novel tool to selectively manipulate the levels of secondary isoprenoids by targeting their precursor supply.

Cation‐ and S ‐adenosyl‐l ‐methionine (AdoMet)‐dependent plant natural product methyltransferases are referred to as CCoAOMTs because of their preferred substrate, caffeoyl coenzyme A (CCoA). The enzymes are encoded by a small family of genes, some of which with a proven role in lignin monomer biosynthesis. In Arabidopsis thaliana individual members of this gene family are temporally and spatially regulated. The gene At1g67990 is specifically expressed in flower buds, and is not detected in any other organ, such as roots, leaves or stems. Several lines of evidence indicate that the At1g67990 transcript is located in the flower buds, whereas the corresponding CCoAOMT‐like protein, termed AtTSM1, is located exclusively in the tapetum of developing stamen. Flowers of At1g67990 RNAi‐suppressed plants are characterized by a distinct flower chemotype with severely reduced levels of the N  ′,N  ′′‐ bis‐(5‐hydroxyferuloyl)‐N  ′′′‐sinapoylspermidine compensated for by N1 ,N5 ,N10 ‐tris‐(5‐hydroxyferuloyl)spermidine derivative, which is characterized by the lack of a single methyl group in the sinapoyl moiety. This severe change is consistent with the observed product profile of AtTSM1 for aromatic phenylpropanoids. Heterologous expression of the recombinant protein shows the highest activity towards a series of caffeic acid esters, but 5‐hydroxyferuloyl spermidine conjugates are also accepted substrates. The in vitro substrate specificity and the in vivo RNAi‐mediated suppression data of the corresponding gene suggest a role of this cation‐dependent CCoAOMT‐like protein in the stamen/pollen development of A. thaliana .

The putative two‐pore Ca2+ channel TPC1 has been suggested to be involved in responses to abiotic and biotic stresses. We show that AtTPC1 co‐localizes with the K+‐selective channel AtTPK1 in the vacuolar membrane. Loss of AtTPC1 abolished Ca2+‐activated slow vacuolar (SV) currents, which were increased in AtTPC1 ‐over‐expressing Arabidopsis compared to the wild‐type. A Ca2+‐insensitive vacuolar cation channel, as yet uncharacterized, could be resolved in tpc1‐2 knockout plants. The kinetics of ABA‐ and CO2‐induced stomatal closure were similar in wild‐type and tpc1‐2 knockout plants, excluding a role of SV channels in guard‐cell signalling in response to these physiological stimuli. ABA‐, K+‐, and Ca2+‐dependent root growth phenotypes were not changed in tpc1‐2 compared to wild‐type plants. Given the permeability of SV channels to mono‐ and divalent cations, the question arises as to whether TPC1 in vivo represents a pathway for Ca2+ entry into the cytosol. Ca2+ responses as measured in aequorin‐expressing wild‐type, tpc1‐2 knockout and TPC1 ‐over‐expressing plants disprove a contribution of TPC1 to any of the stimulus‐induced Ca2+ signals tested, including abiotic stresses (cold, hyperosmotic, salt and oxidative), elevation in extracellular Ca2+ concentration and biotic factors (elf18, flg22). In good agreement, stimulus‐ and Ca2+‐dependent gene activation was not affected by alterations in TPC1 expression. Together with our finding that the loss of TPC1 did not change the activity of hyperpolarization‐activated Ca2+‐permeable channels in the plasma membrane, we conclude that TPC1, under physiological conditions, functions as a vacuolar cation channel without a major impact on cytosolic Ca2+ homeostasis.

In plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two‐hybrid screening we have found three previously uncharacterized receptor‐like protein kinases to be Rop GTPase‐interacting molecules: a cysteine‐rich receptor kinase, named NCRK, and two receptor‐like cytosolic kinases from the Arabidopsis RLCK‐VIb family, named RBK1 and RBK2. Uniquely for Rho‐family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP‐bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro , suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co‐localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single‐copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK‐VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2 . We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross‐talk between Rop GTPases and specific receptor‐like kinases through direct molecular interaction.

Acridone alkaloids formed by acridone synthase in Ruta graveolens L. are composed of N ‐methylanthraniloyl CoA and malonyl CoAs. A 1095 bp cDNA from elicited Ruta cells was expressed in Escherichia coli , and shown to encode S‐ adenosyl‐l ‐methionine‐dependent anthranilate N ‐methyltransferase. SDS–PAGE of the purified enzyme revealed a mass of 40 ± 2 kDa, corresponding to 40 059 Da for the translated polypeptide, whereas the catalytic activity was assigned to a homodimer. Alignments revealed closest relationships to catechol or caffeate O ‐methyltransferases at 56% and 55% identity (73% similarity), respectively, with little similarity (∼20%) to N ‐methyltransferases for purines, putrescine, glycine, or nicotinic acid substrates. Notably, a single Asn residue replacing Glu that is conserved in caffeate O ‐methyltransferases determines the catalytic efficiency. The recombinant enzyme showed narrow specificity for anthranilate, and did not methylate catechol, salicylate, caffeate, or 3‐ and 4‐aminobenzoate. Moreover, anthraniloyl CoA was not accepted. As Ruta graveolens acridone synthase also does not accept anthraniloyl CoA as a starter substrate, the anthranilate N ‐methylation prior to CoA activation is a key step in acridone alkaloid formation, channelling anthranilate from primary into secondary branch pathways, and holds promise for biotechnological applications. RT‐PCR amplifications and Western blotting revealed expression of the N ‐methyltransferase in all organs of Ruta plants, particularly in the flower and root, mainly associated with vascular tissues. This expression correlated with the pattern reported previously for expression of acridone synthase and acridone alkaloid accumulation.