Glyceollin Transcription Factor GmMYB29A2 Regulates Soybean Resistance to Phytophthora sojae.

Glyceollin isomers I, II, and III are the major pathogen-elicited secondary metabolites (i.e. phytoalexins) of soybean (Glycine max) that, collectively with other 5-deoxyisoflavonoids, provide race-specific resistance to Phytophthora sojae. The NAC-family transcription factor (TF) GmNAC42-1 is an essential regulator of some but not all glyceollin biosynthesis genes, indicating other essential TF(s) of the glyceollin gene regulatory network remain to be identified. Here, we conducted comparative transcriptomics on soybean hairy roots of the variety Williams 82 and imbibing seeds of Harosoy 63 upon treatment with wall glucan elicitor from P. sojae and identified two homologous R2R3-type MYB TF genes, GmMYB29A1 and GmMYB29A2, up-regulated during the times of peak glyceollin biosynthesis. Overexpression and RNA interference silencing of GmMYB29A2 increased and decreased expression of GmNAC42-1, GmMYB29A1, and glyceollin biosynthesis genes and metabolites, respectively, in response to wall glucan elicitor. By contrast, overexpressing or silencing GmMYB29A1 decreased glyceollin I accumulation with marginal or no effects on the expressions of glyceollin synthesis genes, suggesting a preferential role in promoting glyceollin turnover and/or competing biosynthetic pathways. GmMYB29A2 interacted with the promoters of two glyceollin I biosynthesis genes in vitro and in vivo. Silencing GmMYB29A2 in Williams 82, a soybean variety that encodes the resistance gene Rps1k, rendered it compatible with race 1 P. sojae, whereas overexpressing GmMYB29A2 rendered the susceptible Williams variety incompatible. Compatibility and incompatibility coincided with reduced and enhanced accumulations of glyceollin I but not other 5-deoxyisoflavonoids. Thus, GmMYB29A2 is essential for accumulation of glyceollin I and expression of Phytophthora resistance.

The NAC family transcription factor GmNAC42-1 regulates biosynthesis of the anticancer and neuroprotective glyceollins in soybean.

BACKGROUND: Glyceollins are isoflavonoid-derived pathogen-inducible defense metabolites (phytoalexins) from soybean (Glycine max L. Merr) that have important roles in providing defense against pathogens. They also have impressive anticancer and neuroprotective activities in mammals. Despite their potential usefulness as therapeutics, glyceollins are not economical to synthesize and are biosynthesized only transiently and in low amounts in response to specific stresses. Engineering the regulation of glyceollin biosynthesis may be a promising approach to enhance their bioproduction, yet the transcription factors (TFs) that regulate their biosynthesis have remained elusive. To address this, we first aimed to identify novel abiotic stresses that enhance or suppress the elicitation of glyceollins and then used a comparative transcriptomics approach to search for TF gene candidates that may positively regulate glyceollin biosynthesis. RESULTS: Acidity stress (pH 3.0 medium) and dehydration exerted prolonged (week-long) inductive or suppressive effects on glyceollin biosynthesis, respectively. RNA-seq found that all known biosynthetic genes were oppositely regulated by acidity stress and dehydration, but known isoflavonoid TFs were not. Systemic acquired resistance (SAR) genes were highly enriched in the geneset. We chose to functionally characterize the NAC (NAM/ATAF1/2/CUC2)-family TF GmNAC42-1 that was annotated as an SAR gene and a homolog of the Arabidopsis thaliana (Arabidopsis) indole alkaloid phytoalexin regulator ANAC042. Overexpressing and silencing GmNAC42-1 in elicited soybean hairy roots dramatically enhanced and suppressed the amounts of glyceollin metabolites and biosynthesis gene mRNAs, respectively. Yet, overexpressing GmNAC42-1 in non-elicited hairy roots failed to stimulate the expressions of all biosynthesis genes. Thus, GmNAC42-1 was necessary but not sufficient to activate all biosynthesis genes on its own, suggesting an important role in the glyceollin gene regulatory network (GRN). The GmNAC42-1 protein directly bound the promoters of biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system. CONCLUSIONS: Acidity stress is a novel elicitor and dehydration is a suppressor of glyceollin biosynthesis. The TF gene GmNAC42-1 is an essential positive regulator of glyceollin biosynthesis. Overexpressing GmNAC42-1 in hairy roots can be used to increase glyceollin yields > 10-fold upon elicitation. Thus, manipulating the expressions of glyceollin TFs is an effective strategy for enhancing the bioproduction of glyceollins in soybean.

Excess coenzyme A reduces skeletal muscle performance and strength in mice overexpressing human PANK2.

Coenzyme A (CoA) is a cofactor that is central to energy metabolism and CoA synthesis is controlled by the enzyme pantothenate kinase (PanK). A transgenic mouse strain expressing human PANK2 was derived to determine the physiological impact of PANK overexpression and elevated CoA levels. The Tg(PANK2) mice expressed high levels of the transgene in skeletal muscle and heart; however, CoA was substantially elevated only in skeletal muscle, possibly associated with the comparatively low endogenous levels of acetyl-CoA, a potent feedback inhibitor of PANK2. Tg(PANK2) mice were smaller, had less skeletal muscle mass and displayed significantly impaired exercise tolerance and grip strength. Skeletal myofibers were characterized by centralized nuclei and aberrant mitochondria. Both the content of fully assembled complex I of the electron transport chain and ATP levels were reduced, while markers of oxidative stress were elevated in Tg(PANK2) skeletal muscle. These abnormalities were not detected in the Tg(PANK2) heart muscle, with the exception of spotty loss of cristae organization in the mitochondria. The data demonstrate that excessively high CoA may be detrimental to skeletal muscle function.

Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation.

BACKGROUND: The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (Panicum virgatum L.) ubiquitin genes (PvUbi1 and PvUbi2) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. PvUbi1 and PvUbi2 promoters) fused to the uidA coding region (GUS) and tested for transient and stable expression in a variety of plant species and tissues. RESULTS: PvUbi1 consists of 607 bp containing cis-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. PvUbi2 consists of 692 bp containing cis-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. PvUbi1 and PvUbi2 were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, PvUbi1 and PvUbi2 promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV 35S, 2x35S, ZmUbi1, and OsAct1 promoters. GUS staining following stable transformation in rice demonstrated that the PvUbi1 and PvUbi2 promoters drove expression in all examined tissues. When stably transformed into tobacco (Nicotiana tabacum), the PvUbi2+3 and PvUbi2+9 promoter fusion variants showed expression in vascular and reproductive tissues. CONCLUSIONS: The PvUbi1 and PvUbi2 promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.

Genetic diversity in Hypericum and AFLP Markers for species-specific identification of H. perforatum L.

One of the top-selling medicinal products worldwide is Hypericum perforatum (St. John's Wort). Despite its cosmopolitan distribution and utilization, little is known regarding the relationship of the bioactive compounds in H. perforatum to the plants from which they are purportedly derived. In this study, amplified fragment length polymorphism (AFLP) analysis of 56 Hypericum accessions, representing 11 species, was conducted to gain a better understanding of diversity within Hypericum species, especially within cultivated accessions of H. perforatum, and to establish a molecular methodology that will provide breeders and regulators with a simple, affordable, and accurate tool with which to identify purported H. perforatum material. Utilizing four primer combinations, a total of 298 polymorphic markers were generated, of which 17 were present in all H. perforatum accessions and 2 were specific to only H. perforatum. This study demonstrates that AFLP can be utilized not only to determine the relationships of closely related Hypericum accessions, but as a tool to authenticate material in herbal remedies through the use of genetic fingerprinting.

A novel approach for characterizing expression levels of genes duplicated by polyploidy.

Studying gene expression in polyploids is complicated by genomewide gene duplication and the problem of distinguishing transcript pools derived from each of the two homeologous genomes such as the A- and D-genomes of allotetraploid Gossypium. Short oligonucleotide probes designed to specifically target several hundred homeologous gene pairs of Gossypium were printed on custom NimbleGen microarrays. These results demonstrate that relative expression levels of homeologous genes may be measured by microarrays and that deviation from equal expression levels of homeologous loci may be common in the allotetraploid nucleus of Gossypium.