Peter Kamp Busk

Regulation of abscisic acid-induced transcription

The phytohormone abscisic acid is probably present in all higher plants. This hormone is necessary for regulation of several events during seed development and for the response to environmental stresses such as desiccation, salt and cold. An important part of the physiological response to abscisic acid is achieved through gene expression.Here, we summarize the current knowledge of regulation of abscisic acid-induced transcription. The main focus is on a description of the known abscisic acid-responsive cis-elements, their properties and the possible transacting factors binding to the elements. Results have shown that cooperative action of cis-elements and the promoter configuraton is crucial for regulation by abscisic acid. Furthermore, several elements are organ- and species-specific. Recent studies of the chromatin structure of abscisic acid-responsive genes point to the importance of induction of transcription by coactivators or by phosphorylation/dephosphorylation of transcription factors. An interesting example of activation by a cofactor is the cooperative action between abscisic acid-signaling and the regulatory protein Viviparous 1 through the abscisic acid responsive element.

Cassava Plants with a Depleted Cyanogenic Glucoside Content in Leaves and Tubers. Distribution of Cyanogenic Glucosides, Their Site of Synthesis and Transport, and Blockage of the Biosynthesis by RNA Interference Technology

Transgenic cassava (Manihot esculenta Crantz, cv MCol22) plants with a 92% reduction in cyanogenic glucoside content in tubers and acyanogenic (<1% of wild type) leaves were obtained by RNA interference to block expression of CYP79D1 and CYP79D2, the two paralogous genes encoding the first committed enzymes in linamarin and lotaustralin synthesis. About 180 independent lines with acyanogenic (<1% of wild type) leaves were obtained. Only a few of these were depleted with respect to cyanogenic glucoside content in tubers. In agreement with this observation, girdling experiments demonstrated that cyanogenic glucosides are synthesized in the shoot apex and transported to the root, resulting in a negative concentration gradient basipetal in the plant with the concentration of cyanogenic glucosides being highest in the shoot apex and the petiole of the first unfolded leaf. Supply of nitrogen increased the cyanogenic glucoside concentration in the shoot apex. In situ polymerase chain reaction studies demonstrated that CYP79D1 and CYP79D2 were preferentially expressed in leaf mesophyll cells positioned adjacent to the epidermis. In young petioles, preferential expression was observed in the epidermis, in the two first cortex cell layers, and in the endodermis together with pericycle cells and specific parenchymatic cells around the laticifers. These data demonstrate that it is possible to drastically reduce the linamarin and lotaustralin content in cassava tubers by blockage of cyanogenic glucoside synthesis in leaves and petioles. The reduced flux to the roots of reduced nitrogen in the form of cyanogenic glucosides did not prevent tuber formation.

Dhurrin Synthesis in Sorghum Is Regulated at the Transcriptional Level and Induced by Nitrogen Fertilization in Older Plants

The content of the cyanogenic glucoside dhurrin in sorghum (Sorghum bicolor L. Moench) varies depending on plant age and growth conditions. The cyanide potential is highest shortly after onset of germination. At this stage, nitrogen application has no effect on dhurrin content, whereas in older plants, nitrogen application induces an increase. At all stages, the content of dhurrin correlates well with the activity of the two biosynthetic enzymes, CYP79A1 and CYP71E1, and with the protein and mRNA level for the two enzymes. During development, the activity of CYP79A1 is lower than the activity of CYP71E1, suggesting that CYP79A1 catalyzes the rate-limiting step in dhurrin synthesis as has previously been shown using etiolated seedlings. The site of dhurrin synthesis shifts from leaves to stem during plant development. In combination, the results demonstrate that dhurrin content in sorghum is largely determined by transcriptional regulation of the biosynthetic enzymes CYP79A1 and CYP71E1.

Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

BackgroundMicroRNAs are important regulators of gene expression at the post-transcriptional level and play an important role in many biological processes. Due to the important biological role it is of great interest to quantitatively determine their expression level in different biological settings.ResultsWe describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision in microRNA quantification.ConclusionsMiR-specific quantitative RT-PCR with DNA primers is a highly specific, sensitive and accurate method for microRNA quantification.

Involvement of cyclin D activity in left ventricle hypertrophy in vivo and in vitro

OBJECTIVE Cardiac hypertrophy is induced by a number of stimuli and can lead to cardiomyopathy and heart failure. Present knowledge suggests that cell-cycle regulatory proteins take part in hypertrophy. We have investigated if the D-type cyclins are involved in cardiac hypertrophy. METHODS The expression and activity of the D-type cyclins and associated kinases in cardiomyocytes were studied during angiotensin II- and pressure overload-induced hypertrophy in rats (Rattus norvegicus) and in isolated, neonatal cardiomyocytes. Expression of the D-type cyclins was manipulated pharmacologically and genetically in neonatal myocytes. RESULTS In the left ventricle, there was a low, constitutive expression of the D-type cyclins, which may have a biological role in normal, adult myocytes. The protein level and the associated kinase activity of the D-type cyclins were up-regulated during hypertrophic growth. The increase in cyclin D expression could be mimicked in vitro in neonatal cardiac myocytes. Interestingly, the cyclin Ds were up-regulated by hypertrophic elicitors that stimulate different signalling pathways, suggesting that cyclin D expression is an inherent part of cardiac hypertrophy. Treatment of myocytes with the compound differentiation inducing factor 1 inhibited expression of the D-type cyclins and impaired hypertrophic growth induced by angiotensin II, phenylephrine and serum. The response to hypertrophic elicitors could be restored in differentiation inducing factor 1-treated myocytes by expressing cyclin D2 from a heterologous promoter. CONCLUSION Our results point to the D-type cyclins as important regulators of cardiac hypertrophy. This supports the notion that cell-cycle regulatory proteins regulate hypertrophic growth.

A tool for design of primers for microRNA-specific quantitative RT-qPCR

BackgroundMicroRNAs are small but biologically important RNA molecules. Although different methods can be used for quantification of microRNAs, quantitative PCR is regarded as the reference that is used to validate other methods. Several commercial qPCR assays are available but they often come at a high price and the sequences of the primers are not disclosed. An alternative to commercial assays is to manually design primers but this work is tedious and, hence, not practical for the design of primers for a larger number of targets.ResultsI have developed the software miRprimer for automatic design of primers for the method miR-specific RT-qPCR, which is one of the best performing microRNA qPCR methods available. The algorithm is based on an implementation of the previously published rules for manual design of miR-specific primers with the additional feature of evaluating the propensity of formation of secondary structures and primer dimers. Testing of the primers showed that 76 out of 79 primers (96%) worked for quantification of microRNAs by miR-specific RT-qPCR of mammalian RNA samples. This success rate corresponds to the success rate of manual primer design. Furthermore, primers designed by this method have been distributed to several labs and used successfully in published studies.ConclusionsThe software miRprimer is an automatic and easy method for design of functional primers for miR-specific RT-qPCR. The application is available as stand-alone software that will work on the MS Windows platform and in a developer version written in the Ruby programming language.

Isolation and Functional Characterisation of Two New bZIP Maize Regulators of the ABA Responsive Gene rab28

The plant hormone abscisic acid regulates gene expression in response to growth stimuli and abiotic stress. Previous studies have implicated members of the bZIP family of transcription factors as mediators of abscisic acid dependent gene expression through the ABRE cis-element. Here, we identify two new maize bZIP transcription factors, EmBP-2 and ZmBZ-1 related to EmBP-1 and OsBZ-8 families. They are differentially expressed during embryo development; EmBP-2 is constitutive, whereas ZmBZ-1 is abscisic acid-inducible and accumulates during late embryogenesis. Both factors are nuclear proteins that bind to ABREs and activate transcription of the abscisic acid-inducible gene rab28 from maize. EmBP-2 and ZmBZ-1 are phosphorylated by protein kinase CK2 and phosphorylation alters their DNA binding properties. Our data suggest that EmBP-2 and ZmBZ-1 are involved in the expression of abscisic acid inducible genes such as rab28 and their activity is modulated by ABA and by phosphorylation.

MicroRNA Expression Profiling of the Porcine Developing Brain

Background MicroRNAs are small, non-coding RNA molecules that regulate gene expression at the post-transcriptional level and play an important role in the control of developmental and physiological processes. In particular, the developing brain contains an impressive diversity of microRNAs. Most microRNA expression profiling studies have been performed in human or rodents and relatively limited knowledge exists in other mammalian species. The domestic pig is considered to be an excellent, alternate, large mammal model for human-related neurological studies, due to its similarity in both brain development and the growth curve when compared to humans. Considering these similarities, studies examining microRNA expression during porcine brain development could potentially be used to predict the expression profile and role of microRNAs in the human brain. Methodology/Principal Findings MicroRNA expression profiling by use of microRNA microarrays and qPCR was performed on the porcine developing brain. Our results show that microRNA expression is regulated in a developmentally stage-specific, as well as a tissue-specific manner. Numerous developmental stage or tissue-specific microRNAs including, miR-17, miR-18a, miR-29c, miR-106a, miR-135a and b, miR-221 and miR-222 were found by microarray analysis. Expression profiles of selected candidates were confirmed by qPCR. Conclusions/Significance The differential expression of specific microRNAs in fetal versus postnatal samples suggests that they likely play an important role in the regulation of developmental and physiological processes during brain development. The data presented here supports the notion that microRNAs act as post-transcriptional switches which may regulate gene expression when required.

MicroRNA profiling in early hypertrophic growth of the left ventricle in rats

Pressure overload induces hypertrophic growth of the heart and in the long term this condition can lead to cardiomyopathy and heart failure. Several miRNAs are upregulated in heart failure. However, it is not clear, which miRNAs (if any) are induced during the early hypertrophic growth phase. To investigate whether the upregulation of miRNAs is an integrated part of hypertrophic growth or an effect of cardiac disease we investigated miRNA expression in early hypertrophic development. Hypertrophy was induced by banding of the ascending aorta of male rats. After 14 days, the heart left ventricle weight relative to body weight of animals with aortic banding had increased 65% compared to matched control rats. Furthermore, RNA was extracted from left ventricles and reverse transcription qPCR showed that expression of the hypertrophic markers atrial natriuretic peptide and brain natriuretic peptide was highly induced in animals with aortic banding. Out of 13 miRs that have previously been reported to be associated with late-stage pressure-overload-induced hypertrophy and heart failure only four (miR-23a, miR-27b, miR-125b and miR-195) were induced during early hypertrophic growth. These miRs were previously associated with angiogenesis and cell growth and their expression in early hypertrophic growth was accompanied by a twofold upregulation of the cell-cycle regulator cyclin D2 that is a marker of cardiac growth. Our results indicate that different miRNAs are involved in early hypertrophic growth than in late stage pressure-overload induced heart failure.

PROTEIN BINDING TO THE ABSCISIC ACID-RESPONSIVE ELEMENT IS INDEPENDENT OF VIVIPAROUS1 IN VIVO

The plant hormone abscisic acid and the transcriptional activator VIVIPAROUS1 have a synergistic effect on transcription during embryo development. An abscisic acid-responsive element (ABRE) mediates induction by abscisic acid and VIVIPAROUS1, but the mechanism involved has not been determined. In this study, we explore the interaction between abscisic acid and VIVIPAROUS1 and its effect on the ABRE from the maize rab28 gene. In transient transformation experiments, abscisic acid stimulated transcription via several elements, whereas activation by VIVIPAROUS1 was mediated exclusively through the ABRE. In vivo footprinting showed only minor differences in binding to the ABRE between wild-type and VIVIPAROUS1-deficient embryos, suggesting that VIVIPAROUS1 stimulates transcription through the ABRE without major changes in protein-DNA interactions. A factor that bound to the ABRE in electrophoretic mobility shift assays was present at the same developmental stages as rab28 mRNA and had binding characteristics similar to those observed by in vivo footprinting. This suggests that the factor binds to the ABRE in the rab28 promoter in vivo. We discuss the constraints that our results put on the possible mechanism for action of VIVIPAROUS1 in vivo.