Carsten Müssig

12-Oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis

Abstract. In addition to OPR1 and OPR2, two isoenzymes of 12-oxophytodienoate reductase, a third isoform (OPR3) has recently been identified in Arabidopsis thaliana (L.) Heynh. The expression of the OPR3 gene is induced not only by a variety of stimuli, such as touch, wind, wounding, UV-light and application of detergent, but also by brassinosteroids. The three enzymes were expressed in a functional form in Escherichia coli, and OPR2 was additionally expressed in insect cell cultures and overexpressed in A. thaliana. Substrate conversion was analyzed using a stereospecific assay. The results show that OPR3 effectively converts the natural (9S,13S)-12-oxophytodienoic acid [Km = 35 μM, Vmax 53.7 nkat (mg protein)−1] to the corresponding 3-2(2′(Z)-pentenyl) cyclopentane-1-octanoic acid (OPC-8:0) stereoisomer while OPR1 and OPR2 convert (9S,13S)-12-oxophytodienoic acid with greatly reduced efficiency compared to OPR3. Thus, OPR3 is the isoenzyme relevant for jasmonate biosynthesis.

Brassinosteroid-Regulated Gene Expression

Major brassinosteroid (BR) effects such as BR-induced growth are mediated through genomic pathways because RNA synthesis inhibitors and protein synthesis inhibitors interfere with these processes. A limited number of BR-regulated genes have been identified hitherto. The majority of genes (such as BRU1, CycD3,Lin6, OPR3, and TRIP-1) were identified by comparisons of BR-treated versus control-treated plants. However, altered transcript levels after BR application may not reflect normal physiological events. A complementary approach is the comparison of BR-deficient plants versus wild-type plants. No artificial treatments interfere with endogenous signaling pathways, but a subset of phenotypic alterations of phytohormone-deficient plants most probably is secondary. To identify genes that are subject to direct BR regulation, we analyzed CPD antisense anddwf1-6 (cbb1) mutant plants. Both show a mild phenotype in comparison with BR-deficient mutants such ascpd/cbb3, det2, anddwf4. Plants were grown under two different environments to filter out BR deficiency effects that occur only at certain environmental conditions. Finally, we established expression patterns after BR treatment of wild-type and dwf1-6(cbb1) plants. Ideally, a BR-regulated gene displays a dose-response relationship in such a way that a gene with decreased transcript levels in BR-deficient plants is BR inducible and vice versa. Expression profile analysis of above ground part of plants was performed by means of Affymetrix Arabidopsis Genome Arrays.

Brassinosteroids Promote Root Growth in Arabidopsis

Although brassinosteroids (BRs) are known to regulate shoot growth, their role in the regulation of root growth is less clear. We show that low concentrations of BRs such as 24-epicastasterone and 24-epibrassinolide promote root elongation in Arabidopsis wild-type plants up to 50% and in BR-deficient mutants such as dwf1-6 (cbb1) and cbb3 (which is allelic to cpd) up to 150%. The growth-stimulating effect of exogenous BRs is not reduced by the auxin transport inhibitor 2,3,5-triidobenzoic acid. BR-deficient mutants show normal gravitropism, and 2,3,5-triidobenzoic acid or higher concentrations of 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid inhibit root growth in the mutants to the same extent as in wild-type plants. Simultaneous administration of 24-epibrassinolide and 2,4-dichlorophenoxyacetic acid results in largely additive effects. Exogenous gibberellins do not promote root elongation in the BR-deficient mutants, and the sensitivity to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid is not altered. Thus, the root growth-stimulating effect of BRs appears to be largely independent of auxin and gibberellin action. Furthermore, we analyzed BR interactions with other phytohormones on the gene expression level. Only a limited set of auxin- and ethylene-related genes showed altered expression levels. Genes related to other phytohormones barely showed changes, providing further evidence for an autonomous stimulatory effect of BR on root growth.

A novel stress-inducible 12-oxophytodienoate reductase from Arabidopsis thaliana provides a potential link between Brassinosteroid-action and Jasmonic-acid synthesis

Summary To isolate brassinosteroid (BR) inducible genes, a subtractive cDNA-cloning strategy was applied. One of the isolated genes encodes a plant homologue to yeast old yellow enzymes (OYE) with strong sequence similarity to two cloned 12-oxo-phytodienoic acid reductases ( OPR1 and OPR2 ) from A. thaliana and was termed 12-oxophytodienoate reductase 3 ( OPR3 ; accession number: AJ238149). The expression of the OPR3 gene is induced by brassinosteroids, jasmonic acid (JA), and by a variety of stimuli like UV-light, touch, wind, wounding, and application of a detergent. Recombinant OPR3 protein converts 12-oxophytodienoate (OPDA) into 12-oxo phytoenoic acid (OPC8: 0), indicating the participation of OPR3 in the biosynthesis of JA from linolenic acid via the Vick-Zimmerman-pathway. In plants, OPC8: 0 is inevitably metabolized to JA by three cycles of β-oxidation. Both OPDA and JA are signal molecules involved in developmental processes and stress responses. Depending on environmental or developmental conditions, OPR potentially regulates the ratio between these two signal molecules. The yeast old yellow enzymes act on various enones and phenols including steroids, catalyzing reduction and disproportionation reactions. Thus, in addition to OPDA to OPC8: 0 conversion, OPR3 might be involved in further biosynthetic or degradative pathways in plants. As OPR3 expression is increased through treatment with brassinosteroids, it provides a potential link between brassinosteroid action and JA synthesis. BRs may thus influence the stress responses of plants through stimulation of JA synthesis.

Identification of brassinosteroid-related genes by means of transcript co-response analyses.

The comprehensive systems-biology database (CSB.DB) was used to reveal brassinosteroid (BR)-related genes from expression profiles based on co-response analyses. Genes exhibiting simultaneous changes in transcript levels are candidates of common transcriptional regulation. Combining numerous different experiments in data matrices allows ruling out outliers and conditional changes of transcript levels. CSB.DB was queried for transcriptional co-responses with the BR-signalling components BRI1 and BAK1: 301 out of 9694 genes represented in the nasc0271 database showed co-responses with both genes. As expected, these genes comprised pathway-involved genes (e.g. 72 BR-induced genes), because the BRI1 and BAK1 proteins are required for BR-responses. But transcript co-response takes the analysis a step further compared with direct approaches because BR-related non BR-responsive genes were identified. Insights into networks and the functional context of genes are provided, because factors determining expression patterns are reflected in correlations. Our findings demonstrate that transcript co-response analysis presents a valuable resource to uncover common regulatory patterns of genes. Different data matrices in CSB.DB allow examination of specific biological questions. All matrices are publicly available through CSB.DB. This work presents one possible roadmap to use the CSB.DB resources.

Physiology and molecular mode of action of brassinosteroids

Abstract Brassinosteroids (BRs) comprise a group of polyhydroxysteroids, which show close structural similarity to steroid hormones from arthropods and mammals. BRs are now accepted as a new class of phytohormones due to their ubiquitous occurrence in plants, their highly effective elicitation of various responses and the identification of mutants defective in BR-biosynthesis or -response. Important steps of BR-biosynthesis were elucidated with precursor-feeding experiments and by the analysis of BR-biosynthesis-deficient mutants. The altered phenotypes of these mutants, particularly in Arabidopsis , revealed the essential nature of BRs for normal growth and development. A major role of BRs is the positive regulation of cell expansion. Furthermore, BRs modulate plant responses to biotic and abiotic stresses and to other phytohormones, and influence differentiation processes of cells and tissues. BR-insensitive mutants such as bri1 hold the potential for uncovering BR-signalling pathway(s) at the molecular level. The identification of BR-regulated genes demonstrates a genetic basis for BR mode of action with reference to their multiple effects. This review focuses on the relevance of BRs to the control of various physiological processes, BR-signalling and underlying molecular mechanisms by considering known mutants.

The AtNFXL1 gene encodes a NF-X1 type zinc finger protein required for growth under salt stress

The human NF‐X1 protein and homologous proteins in eukaryotes represent a class of transcription factors which are characterised by NF‐X1 type zinc finger motifs. The Arabidopsis genome encodes two NF‐X1 homologs, which we termed AtNFXL1 and AtNFXL2. Growth and survival was impaired in atnfxl1 knock‐out mutants and AtNFXL1‐antisense plants under salt stress in comparison to wild‐type plants. In contrast, 35S∷AtNFXL1 plants showed higher survival rates. The AtNFXL2 protein potentially plays an antagonistic role. The Arabidopsis NF‐X1 type zinc finger proteins likely are part of regulatory mechanisms, which protect major processes such as photosynthesis.

The extracellular EXO protein mediates cell expansion in Arabidopsis leaves

BackgroundThe EXO (EXORDIUM) gene was identified as a potential mediator of brassinosteroid (BR)-promoted growth. It is part of a gene family with eight members in Arabidopsis. EXO gene expression is under control of BR, and EXO overexpression promotes shoot and root growth. In this study, the consequences of loss of EXO function are described.ResultsThe exo loss of function mutant showed diminished leaf and root growth and reduced biomass production. Light and scanning electron microscopy analyses revealed that impaired leaf growth is due to reduced cell expansion. Epidermis, palisade, and spongy parenchyma cells were smaller in comparison to the wild-type. The exo mutant showed reduced brassinolide-induced cotyledon and hypocotyl growth. In contrast, exo roots were significantly more sensitive to the inhibitory effect of synthetic brassinolide. Apart from reduced growth, exo did not show severe morphological abnormalities. Gene expression analyses of leaf material identified genes that showed robust EXO-dependent expression. Growth-related genes such as WAK1, EXP5, and KCS1, and genes involved in primary and secondary metabolism showed weaker expression in exo than in wild-type plants. However, the vast majority of BR-regulated genes were normally expressed in exo. HA- and GFP-tagged EXO proteins were targeted to the apoplast.ConclusionThe EXO gene is essential for cell expansion in leaves. Gene expression patterns and growth assays suggest that EXO mediates BR-induced leaf growth. However, EXO does not control BR-levels or BR-sensitivity in the shoot. EXO presumably is involved in a signalling process which coordinates BR-responses with environmental or developmental signals. The hypersensitivity of exo roots to BR suggests that EXO plays a diverse role in the control of BR responses in the root.

EXORDIUM-LIKE1 Promotes Growth during Low Carbon Availability in Arabidopsis

Little is known about genes that control growth and development under low carbon (C) availability. The Arabidopsis (Arabidopsis thaliana) EXORDIUM-LIKE1 (EXL1) gene (At1g35140) was identified as a brassinosteroid-regulated gene in a previous study. We show here that the EXL1 protein is required for adaptation to C- and energy-limiting growth conditions. In-depth analysis of EXL1 transcript levels under various environmental conditions indicated that EXL1 expression is controlled by the C and energy status. Sugar starvation, extended night, and anoxia stress induced EXL1 gene expression. The C status also determined EXL1 protein levels. These results suggested that EXL1 is involved in the C-starvation response. Phenotypic changes of an exl1 loss-of-function mutant became evident only under corresponding experimental conditions. The mutant showed diminished biomass production in a short-day/low-light growth regime, impaired survival during extended night, and impaired survival of anoxia stress. Basic metabolic processes and signaling pathways are presumed to be barely impaired in exl1, because the mutant showed wild-type levels of major sugars, and transcript levels of only a few genes such as QUA-QUINE STARCH were altered. Our data suggest that EXL1 is part of a regulatory pathway that controls growth and development when C and energy supply is poor.

Brassinosteroid signaling in plants

In animals and humans, steroid hormones (SHs) regulate gene transcription via the binding of nuclear receptors. In addition, rapid nongenomic effects of steroids occur and appear to be mediated by plasma-membrane receptors. Plants also use steroids as signaling molecules. These brassinosteroids (BRs) show structural similarity to the SHs of vertebrates and insects. Plant mutants defective in brassinosteroid biosynthesis or perception exhibit dwarfism and reduced fertility, and reveal the need for BRs during growth and development. BR signaling in Arabidopsis thaliana and rice (Oryza sativa) - dicotyledonous and monocotyledonous models, respectively - is mediated by the receptor kinases BRI1 and OsBRI1. The extracellular domain of BRI1 perceives BRs and the signal is mediated via an intracellular kinase domain that autophosphorylates Ser and Thr residues and apparently has the potential to phosphorylate other substrates. BRI1 transduces steroid signals across the plasma membrane and mediates genomic effects.