Tom Hamborg Nielsen

Genome-Wide Analysis of the Arabidopsis Leaf Transcriptome Reveals Interaction of Phosphate and Sugar Metabolism

Global gene expression was analyzed in Arabidopsis (Arabidopsis thaliana) by microarrays comprising 21,500 genes. Leaf segments derived from phosphorus (P)-starved and P-replenished plants were incubated with or without sucrose (Suc) to obtain tissues with contrasting combinations of P and carbohydrate levels. Transcript profiling revealed the influence of the two factors individually and the interactions between P- and sugar-dependent gene regulation. A large number of gene transcripts changed more than 2-fold: In response to P starvation, 171 genes were induced and 16 repressed, whereas Suc incubation resulted in 337 induced and 307 repressed genes. A number of new candidate genes involved in P acquisition were discovered. In addition, several putative transcription factors and signaling proteins of P sensing were disclosed. Several genes previously identified to be sugar responsive were also regulated by P starvation and known P-responsive genes were sugar inducible. Nearly 150 genes were synergistically or antagonistically regulated by the two factors. These genes exhibit more prominent or contrasting regulation in response to Suc and P in combination than expected from the effect of the two factors individually. The genes exhibiting interactions form three main clusters with different response patterns and functionality of genes. One cluster (cluster 1) most likely represents a regulatory program to support increased growth and development when both P and carbohydrates are ample. Another cluster (cluster 3) represents genes induced to alleviate P starvation and these are further induced by carbohydrate accumulation. Thus, interactions between P and Suc reveal two different signaling programs and novel interactions in gene regulation in response to environmental factors. cis-Regulatory elements were analyzed for each factor and for interaction clusters. PHR1 binding sites were more frequent in promoters of P-regulated genes as compared to the entire Arabidopsis genome, and E2F and PHR1 binding sites were more frequent in interaction clusters 1 and 3, respectively.

Starch phosphorylation: a new front line in starch research

Starch is the primary energy reserve in higher plants and is, after cellulose, the second most abundant carbohydrate in the biosphere. It is also the most important energy source in the human diet and, being a biodegradable polymer with well-defined chemical properties, has an enormous potential as a versatile renewable resource. The only naturally occurring covalent modification of starch is phosphorylation. Starch phosphate esters were discovered a century ago but were long regarded as a curiosity, receiving little attention. Indeed, the mechanism for starch phosphorylation remained completely unknown until recently. The starch-phosphorylating enzyme is an alpha-glucan water dikinase. It is now known that starch phosphorylation plays a central role in starch metabolism.

Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity☆

Studies were conducted to determine whether protein phosphorylation may be a mechanism for regulation of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS), shown previously to be light-dark regulated by some type of covalent modification. Radioactive phosphate was incorporated into the 120-kDa subunit of SPS during labeling of excised leaves with [32P]Pi, as shown by immunoprecipitation and denaturing gel electrophoresis of the enzyme. Conditions which activated the enzyme (illumination of leaves or mannose treatment of leaf discs in darkness) reduced the incorporation of radiolabel into SPS in the in vivo system. The partially purified SPS protein could also be phosphorylated in vitro using [gamma-32P]ATP. In the in vitro system, the incorporation of radiolabel into the 120-kDa subunit of SPS was dependent on time and magnesium concentration, and was closely paralleled by inactivation of the enzyme. These results provide the first evidence to establish protein phosphorylation as a mechanism for the covalent regulation of SPS activity.

A [beta]-Amylase in Potato Tubers Is Induced by Storage at Low Temperature

A new starch-degrading enzyme activity is induced by storage of potato (Solanum tuberosum L.) tubers at low temperatures (L. Hill, R. Reimholz, R. Schroder, T.H. Nielsen, M. Stitt [1996] Plant Cell Environ 14: 1223–1237). The cold-induced activity was separated from other amylolytic activities in zymograms based on iodine staining of polyacrylamide gels containing amylopectin. A similar band of activity was detected at normal growth temperatures in leaves, stems, and growing tubers but was present only at low activity in warm-stored tubers. The cold-induced enzyme was separated by ion-exchange chromatography from other amylolytic activities. It has a broad neutral pH optimum. Characterization of its hydrolytic activity with different substrates showed that the cold-induced activity is a [beta]-amylase present at low activity in tubers stored at 20[deg]C and induced progressively when temperatures are decreased to 5 and 3[deg]C. The first clear induction of [beta]-amylase activity was observed within 3 d of storage at 3[deg]C, and the activity increased 4- to 5-fold within 10 d. The possible involvement of the cold-induced [beta]-amylase in sugar accumulation during cold storage is discussed.

Starch Phosphorylation in Potato Tubers Proceeds Concurrently with de Novo Biosynthesis of Starch.

The in vivo phosphorylation of starch was studied in Solanum tuberosum cv Dianella and Posmo. Small starch granules contain 25% more ester-bound phosphate per glucose residue than large starch granules. The degree of phosphorylation was found to be almost constant during tuber development. Isolated tuber discs synthesize starch from externally supplied glucose at a significant rate. Tuber discs supplied with glucose and [32P]orthophosphate incorporate radiolabeled phosphorus into the starch. The level of 32P incorporation is proportional to the amount of starch synthesized. The incorporation of 32P from orthophosphate is correlated to de novo synthesis of starch, since the incorporation of 32P is diminished upon inhibition of starch synthesis by fluoride. Based on the amount of [14C]glucose phosphate isolated after hydrolysis of purified starch from tuber discs incubated in the presence of [U-14C]glucose, approximately 0.5% of the glucose residues of the de novo-synthesized starch are phosphorylated. This value is in general agreement with the observed levels of phosphorus in starch accumulated during tuber development. Thus, the enzyme system responsible for starch phosphorylation is fully active in the isolated tuber discs, and the starch phosphorylation proceeds as an integrated part of de novo starch synthesis.

Dissecting the plant transcriptome and the regulatory responses to phosphate deprivation.

Inorganic phosphate (Pi) is an essential nutrient for plants, and the low bioavailability of Pi in soils is often a limitation to growth and development. Consequently, plants have evolved a range of regulatory mechanisms to adapt to phosphorus-starvation in order to optimise uptake and assimilation of Pi. Recently, significant progress has been made in elucidating these mechanisms. The coordinated expression of a large number of genes is important for many of these adaptations. Several global expression studies using microarray analysis have been conducted in Arabidopsis thaliana. These studies provide a valuable basis for the identification of new regulatory genes and promoter elements to further the understanding of Pi-dependent gene regulation. With focus on the Arabidopsis transcriptome, we extract common findings that point to new groups of putative regulators, including the NAC, MYB, ethylene response factor/APETALA2, zinc-finger, WRKY and CCAAT-binding families. With a number of new discoveries of regulatory elements, a complex regulatory network is emerging. Some regulatory elements, e.g. the transcription factor PHR1 and the microRNA (miRNA) miR399 and associated factors are well documented, yet not fully understood, whereas other suggested components need further characterisation. Here, we evaluate the contribution of the regulatory elements to the P-responses and present a model comprising factors directly or indirectly involved in transcriptional regulation and the role of miRNAs as regulators and long-distance signals. A striking feature is a series of feedback loops and parallel mechanisms that can modify and attenuate responses. We suggest that these mechanisms are instrumental in providing an accurate response and in keeping P-homeostasis.

Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat.

BackgroundGene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.ResultsHere we demonstrate the successful BSMV-mediated virus induced gene silencing (VIGS) of three different genes in barley roots, i.e. the barley homologues of the IPS1, PHR1, and PHO2 genes known to participate in Pi uptake and reallocation in Arabidopsis. Attempts to silence two other genes, the Pi transporter gene HvPht1;1 and the endo-β-1,4-glucanase gene HvCel1, in barley roots were unsuccessful, probably due to instability of the plant gene inserts in the viral vector. In B. distachyon leaves, significant silencing of the PHYTOENE DESATURASE (BdPDS) gene was obtained as shown by photobleaching as well as quantitative RT-PCR analysis. On the other hand, only very limited silencing of the oat AsPDS gene was observed in both hexaploid (A. sativa) and diploid (A. strigosa) oat. Finally, two modifications of the BSMV vector are presented, allowing ligation-free cloning of DNA fragments into the BSMV-γ component.ConclusionsOur results show that BSMV can be used as a vector for gene silencing in barley roots and in B. distachyon leaves and possibly roots, opening up possibilities for using VIGS to study cereal root biology and to exploit the wealth of genome information in the new cereal model plant B. distachyon. On the other hand, the silencing induced by BSMV in oat seemed too weak to be of practical use. The new BSMV vectors modified for ligation-free cloning will allow rapid insertion of plant gene fragments for future experiments.

Intermediary glucan structures formed during starch granule biosynthesis are enriched in short side chains, a dynamic pulse labeling approach

The formation of intermediary glucans, mature starch, and phytoglycogen was studied using leaves of Arabidopsis thaliana wild type and dbe mutant, which lacks plastidic isoamylase (Zeeman, S. C., Umemoto, T., Lue, W. L., Au-Yeung, P., Martin, C., Smith, A. M., and Chen, J. (1998)Plant Cell 10, 1699–1711). A new approach to the study of starch biosynthesis was developed based on “very short pulse” labeling of leaf starch through photosynthetic fixation of14CO2. This allowed selective analysis of the structure of starch formed within a 30-s period. This time frame is shorter than the period required for the formation of a single crystalline amylopectin lamella and consequently permits a direct analysis of intermediary structures during granule formation. Analysis of chain length distribution showed that the most recently formed outer layer of the granules has a structure different from the mature starch. The outer layer is enriched in short chains that are 6–11 glucose residues long. Side chains with 6 glucose residues are the shortest abundant chains formed, and they are formed exclusively by transfer from donor chains of 12 glucose residues or longer. The labeling pattern shows that chain transfer resulting in branching is a rapid and efficient process, and the preferential labeling of shorter chains in the intermediary granule bound glucan is suggested to be a direct consequence of efficient branching. Although similar, the short chain intermediary structure is not identical to phytoglycogen, which is an even more highly branched molecule with very few longer chains (more than 40 glucose residues). Pulse and chase labeling profiles for thedbe mutant showed that the final structure is more highly branched than the intermediary structures, which implies that branching of phytoglycogen occurs over a longer time period than branching of starch.

Global analysis of microRNA in Arabidopsis in response to phosphate starvation as studied by locked nucleic acid‐based microarrays

MicroRNAs (miRNAs) are short RNA chains (20-24 bp) which are emerging as important regulators of gene expression. miRNAs are encoded by specific genes, and in Arabidopsis, 190 genes have presently been identified. It has been shown that miR399 is essential for the phosphate starvation response, and recent studies have shown transcriptional changes in a number of additional miRNAs in response to a shortage of phosphate. In this study, global profiles of the miRNA in shoots of Arabidopsis plants grown on limited phosphate or full nutrient in combination with sucrose feed were analysed using the miRCURY LNA microRNA Array system. Furthermore, changes in miRNA transcript were compared between a mutant lacking the transcription factor phosphate starvation responses 1 (PHR1) and wild-type plants. The global analysis identified miRNAs belonging to nine families to respond to P deprivation, sucrose or PHR1. Among these, miR399d, miR827, miR866, miR391 and miR163 were most prominently induced upon P starvation, whereas miR169b/c was strongly induced in previously starved plants when provided with sufficient P and more so when combined with an addition of sucrose. This study shows that array analysis is in general agreement with data obtained by other high-throughput technologies. The array data were confirmed by real-time reverse transcriptase-polymerase chain reaction analyses of selected pri-miRNAs. Our data corroborate the implication that several miRNAs are involved in the P-starvation response and further identify miR866 and miR163 as new candidates of miRNAs associated with the regulation of the P-starvation response.

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.) : I. Spatial distribution of endogenous cytokinins in relation to leaf growth.

Immunoaffinity purification of zeatin, dihydrozeatin and isopentenyl-type cytokinins from expanding leaves of sweet pepper was accomplished using a single immobilized monoclonal antibody. Isopentenyl adenosine, zeatin, zeatin riboside and the N9-glucosides of zeatin and isopentenyl adenine were found to be the dominating endogenous cytokinins while the dihydrozeatin cytokinins were either absent or constituted a very minor group of cytokinin metabolites in pepper. Leaves were selected for analysis at an age where a range of developmental stages exist within a single leaf. The spatial distribution of endogenous cytokinins in rapidly expanding leaves at this stage was markedly different from the almost uniform distribution in expanded leaves. The distribution of zeatin and zeatin riboside in rapidly expanding leaves was found to be correlated with the rate of leaf expansion which is high (∼40%/24 h) in the basal leaf tissue and low (∼10%/24 h) near the leaf tip. Applied growtn factors supported a rate of expansion of excised discs comparable to the growth rates observed in situ, but did not affect the ability of the tissue to retain assimilated amino acids. The results are discussed in relation to sink-strength stimultation as a potential mode of cytokinin action in leaf development.