Analysis of the MYB28, MYB29 and MYB76 transcription factors involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana

Glucosinolates (GSL) are nitrogen- and sulphur-rich natural plant products that serve as chemoprotective compounds in plant biotic defence reactions against herbivores and pathogens. GS also function as flavour compounds and exhibit strong anticancerogenic properties beneficial to human health. Although considerable progress has been made concerning the biosynthesis of glucosinolates, little is known how plants regulate the synthesis of these metabolites. The MYB28, MYB29 and MYB76 (referred to as HIGH ALIPHATIC GLUCOSINOLATE 1, 3 and 2) transcription factors were identified as novel regulators of glucosinolate biosynthesis. Molecular and biochemical characterization of Arabidopsis gain- and loss-of-function mutants revealed a significant correlation between the MYB28/HAG1, MYB29/HAG3 and MYB76/HAG2 transcript levels and the accumulation of aliphatic Met-derived glucosinolates. MYB28/HAG1, MYB29/HAG3 and MYB76/HAG2 over-expression caused a considerable increase in the level of aliphatic glucosinolates due to the specific activation of genes involved in aliphatic glucosinolate biosynthesis. Disruption of MYB28/HAG1 and MYB29/HAG3 gene functions caused a dramatic decrease in the content of aliphatic glucosinolates, whereas myb76/hag2 loss-of-function mutants showed no changes in glucosinolate profiles except for the slight decrease in the level of 4MSOB glucosinolate. Analysis of the ProHAG:GUS activity revealed similar expression patterns in generative organs and rosette leaves of Arabidopsis plants, covering the main sites of aliphatic glucosinolate accumulation and overlapping with the expression of glucosinolate biosynthetic genes. Mechanical stimuli transiently induced MYB/HAG expression demonstrating their role in early plant responses to biotic stresses. Expression of MYB28/HAG1 was clearly induced by glucose, indicating a novel signaling mechanism for the integration of carbohydrate availability in glucosinolates production, whereas MYB29/HAG3 was shown to be involved in MeJa-induced glucosinolate biosynthesis. Notably, MYB76/HAG2 expression was independent from plant elicitors and seems to play an accessory role in glucosinolate biosynthesis. Besides, MYB28/HAG1 over-expression reduced performance of the generalist lepidopteran herbivore Spodoptera exigua in weight-gain experiments. Finally, MYB28/HAG1, MYB76/HAG2 and MYB29/HAG3 reciprocally trans-activate each other and comprise a complex regulatory network in concert with other regulators (MYB51, MYB34, MYB122, WRKY25 and SLIM1) to control glucosinolate biosynthesis in response to different environmental stimuli

Arabidopsis SWI/SNF chromatin remodeling complex binds both promoters and terminators to regulate gene expression

ATP-dependent chromatin remodeling complexes are important regulators of gene expression in Eukaryotes. In plants, SWI/SNF-type complexes have been shown critical for transcriptional control of key developmental processes, growth and stress responses. To gain insight into mechanisms underlying these roles, we performed whole genome mapping of the SWI/SNF catalytic subunit BRM in Arabidopsis thaliana, combined with transcript profiling experiments. Our data showthatBRM occupies thousands of sites in Arabidopsis genome, most of which located within or close to genes. Among identified direct BRM transcriptional targets almost equal numbers were up- and downregulated upon BRM depletion, suggesting that BRM can act as both activator and repressor of gene expression. Interestingly, in addition to genes showing canonical pattern of BRM enrichment near transcription start site, many other genes showed a transcription termination sitecentred BRM occupancy profile. We found that BRMbound 3� gene regions have promoter-like features, including presence of TATA boxes and high H3K4me3 levels, and possess high antisense transcriptional activity which is subjected to both activation and repression by SWI/SNF complex. Our data suggest that binding to gene terminators and controlling transcription of non-coding RNAs is another way through which SWI/SNF complex regulates expression of its targets

A simplified method for the analysis of transcription factor-promoter interactions that allows high-throughput data generation

Berger B, Stracke R, Yatusevich R, Weisshaar B, Fluegge U-I, Gigolashvili T. A simplified method for the analysis of transcription factor-promoter interactions that allows high-throughput data generation. The Plant Journal. 2007;50(5):911-916

Promoter‐pervasive transcription causes RNA polymerase II pausing to boost DOG1 expression in response to salt

Eukaryotic genomes are pervasively transcribed by RNA polymerase II. Yet, the molecular and biological implications of such a phenomenon are still largely puzzling. Here, we describe noncoding RNA transcription upstream of the Arabidopsis thaliana DOG1 gene, which governs salt stress responses and is a key regulator of seed dormancy. We find that expression of the DOG1 gene is induced by salt stress, thereby causing a delay in seed germination. We uncover extensive transcriptional activity on the promoter of the DOG1 gene, which produces a variety of lncRNAs. These lncRNAs, named PUPPIES, are co-directionally transcribed and extend into the DOG1 coding region. We show that PUPPIES RNAs respond to salt stress and boost DOG1 expression, resulting in delayed germination. This positive role of pervasive PUPPIES transcription on DOG1 gene expression is associated with augmented pausing of RNA polymerase II, slower transcription and higher transcriptional burst size. These findings highlight the positive role of upstream co-directional transcription in controlling transcriptional dynamics of downstream genes

HAG2/MYB76 and HAG3/MYB29 exert a specific and coordinated control on the regulation of aliphatic glucosinolate biosynthesis in *Arabidopsis thaliana*

Gigolashvili T, Engqvist M, Yatusevich R, Müller C, Fluegge U-I. HAG2/MYB76 and HAG3/MYB29 exert a specific and coordinated control on the regulation of aliphatic glucosinolate biosynthesis in *Arabidopsis thaliana*. NEW PHYTOLOGIST. 2008;177(3):627-642.In a previous transactivation screen, two Arabidopsis thaliana R2R3-MYB transcription factors, HAG2/MYB76 and HAG3/MYB29, along with the already characterized HAG1/MYB28, were identified as putative regulators of aliphatic glucosinolate biosynthesis. Molecular and biochemical characterization of HAG2/MYB76 and HAG3/MYB29 functions was performed using transformants with increased or repressed transcript levels. Real-time PCR assays, cotransformation assays and measurements of glucosinolate contents were used to assess the impact of both MYB factors on the steady-state level of glucosinolate biosynthetic genes and accumulation of aliphatic glucosinolates. Both HAG2/MYB76 and HAG3/MYB29 were shown to be positive regulators of aliphatic glucosinolate biosynthesis. Expression of promoter-beta-glucuronidase (GUS) fusions indicated GUS activities in both vegetative and generative organs, with distinct characteristics for each MYB factor. HAG1/MYB28, HAG2/MYB76 and HAG3/MYB29 reciprocally transactivated each other in the control of aliphatic glucosinolate biosynthesis and downregulated the expression of genes involved in the control of indolic glucosinolate biosynthesis, pointing to a reciprocal negative regulation of these two pathways. All three HAG transcription factors exert a coordinated control on aliphatic glucosinolate biosynthesis

The R2R3-MYB transcription factor HAG1/MYB28 is a regulator of methionine-derived glucosinolate biosynthesis in Arabidopsis thaliana

Methionine-derived glucosinolates belong to a class of plant secondary metabolites that serve as chemoprotective compounds in plant biotic defense reactions and also exhibit strong anticancerogenic properties beneficial to human health. In a screen for the trans-activation potential of various transcription factors toward glucosinolate biosynthetic genes, we could identify the HAG1 (HIGH ALIPHATIC GLUCOSINOLATE 1, also referred to as MYB28) gene as a positive regulator of aliphatic methionine-derived glucosinolates. The content of aliphatic glucosinolates as well as transcript levels of aliphatic glucosinolate biosynthetic genes were elevated in gain-of-function mutants and decreased in HAG1 RNAi knock-down mutants. ProHAG1:GUS expression analysis revealed strong HAG1 promoter activity in generative organs and mature leaves of A. thaliana plants, the main sites of accumulation of aliphatic glucosinolates. Mechanical stimuli such as touch or wounding transiently induced HAG1/MYB28 expression in inflorescences of flowering plants, and HAG1/MYB28 over-expression reduced insect performance as revealed by weight gain assays with the generalist lepidopteran herbivore Spodoptera exigua. Expression of HAG1/MYB28 was significantly induced by glucose, indicating a novel transcriptional regulatory mechanism for the integration of carbohydrate availability upon biotic challenge. We hypothesize that HAG1/MYB28 is a novel regulator of aliphatic glucosinolate biosynthesis that controls the response to biotic challenges.Tamara Gigolashvili, Ruslan Yatusevich, Bettina Berger, Caroline Müller and Ulf-Ingo Flügg