Aleksandra Skirycz

The DOF transcription factor OBP1 is involved in cell cycle regulation in Arabidopsis thaliana

In contrast to animal growth, plant growth is largely post-embryonic. Therefore plants have developed new mechanisms to precisely regulate cell proliferation by means of internal and external stimuli whilst the general core cell cycle machinery is conserved between eukaryotes. In this work we demonstrate a role for the Arabidopsis thaliana DNA-binding-with-one-finger (DOF) transcription factor OBP1 in the control of cell division upon developmental signalling. Inducible overexpression of OBP1 resulted in a significant overrepresentation of cell cycle genes among the upregulated transcripts. Direct targets of OBP1, as verified by chromatin immunoprecipitation, include at least the core cell cycle gene CYCD3;3 and the replication-specific transcription factor gene AtDOF2;3. Consistent with our molecular data, short-term activation of OBP1 in cell cultures affected cell cycle re-entry, shortening the duration of the G(1) phase and the overall length of the cell cycle, whilst constitutive overexpression of OBP1 in plants influenced cell size and cell number, leading to a dwarfish phenotype. Expression during embryogenesis, germination and lateral root initiation suggests an important role for OBP1 in cell cycle re-entry, operating as a transcriptional regulator of key cell cycle genes. Our findings provide significant input into our understanding of how cell cycle activity is incorporated into plant growth and development.

Oil palm natural diversity and the potential for yield improvement

African oil palm has the highest productivity amongst cultivated oleaginous crops. Species can constitute a single crop capable to fulfill the growing global demand for vegetable oils, which is estimated to reach 240 million tons by 2050. Two types of vegetable oil are extracted from the palm fruit on commercial scale. The crude palm oil and kernel palm oil have different fatty acid profiles, which increases versatility of the crop in industrial applications. Plantations of the current varieties have economic life-span around 25–30 years and produce fruits around the year. Thus, predictable annual palm oil supply enables marketing plans and adjustments in line with the economic forecasts. Oil palm cultivation is one of the most profitable land uses in the humid tropics. Oil palm fruits are the richest plant source of pro-vitamin A and vitamin E. Hence, crop both alleviates poverty, and could provide a simple practical solution to eliminate global pro-vitamin A deficiency. Oil palm is a perennial, evergreen tree adapted to cultivation in biodiversity rich equatorial land areas. The growing demand for the palm oil threatens the future of the rain forests and has a large negative impact on biodiversity. Plant science faces three major challenges to make oil palm the key element of building the future sustainable world. The global average yield of 3.5 tons of oil per hectare (t) should be raised to the full yield potential estimated at 11–18t. The tree architecture must be changed to lower labor intensity and improve mechanization of the harvest. Oil composition should be tailored to the evolving needs of the food, oleochemical and fuel industries. The release of the oil palm reference genome sequence in 2013 was the key step toward this goal. The molecular bases of agronomically important traits can be and are beginning to be understood at the single base pair resolution, enabling gene-centered breeding and engineering of this remarkable crop.

Canga biodiversity, a matter of mining

Brazilian name canga refers to the ecosystems associated with superficial iron crusts typical for the Brazilian state of Minas Gerais (MG) and some parts of Amazon (Flona de Carajas). Iron stone is associated with mountain plateaux and so, in addition to high metal concentrations (particularly iron and manganese), canga ecosystems, as other rock outcrops, are characterized by isolation and environmental harshness. Canga inselbergs, all together, occupy no more than 200 km2 of area spread over thousands of km2 of the Iron Quadrangle (MG) and the Flona de Carajas, resulting in considerable beta biodiversity. Moreover, the presence of different microhabitats within the iron crust is associated with high alpha biodiversity. Hundreds of angiosperm species have been reported so far across remote canga inselbergs and different micro-habitats. Among these are endemics such as the cactus Arthrocereus glaziovii and the medicinal plant Pilocarpus microphyllus. Canga is also home to iron and manganese metallophytes; species that evolved to tolerate high metal concentrations. These are particularly interesting to study metal homeostasis as both iron and manganese are essential plant micro-elements. Besides being models for metal metabolism, metallophytes can be used for bio-remediation of metal contaminated sites, and as such are considered among priority species for canga restoration. “Biodiversity mining” is not the only mining business attracted to canga. Open cast iron mining generates as much as 5–6% of Brazilian gross domestic product and dialog between mining companies, government, society, and ecologists, enforced by legal regulation, is ongoing to find compromise for canga protection, and where mining is unavoidable for ecosystem restoration. Environmental factors that shaped canga vegetation, canga biodiversity, physiological mechanisms to play a role, and ways to protect and restore canga will be reviewed.

Expression of human dopamine receptor in potato (Solanum tuberosum) results in altered tuber carbon metabolism

BackgroundEven though the catecholamines (dopamine, norepinephrine and epinephrine) have been detected in plants their role is poorly documented. Correlations between norepinephrine, soluble sugars and starch concentration have been recently reported for potato plants over-expressing tyrosine decarboxylase, the enzyme mediating the first step of catecholamine synthesis. More recently norepinephrine level was shown to significantly increase after osmotic stress, abscisic acid treatment and wounding. Therefore, it is possible that catecholamines might play a role in plant stress responses by modulating primary carbon metabolism, possibly by a mechanism similar to that in animal cells. Since to date no catecholamine receptor has been identified in plants we transformed potato plants with a cDNA encoding human dopamine receptor (HD1).ResultsTuber analysis of transgenic plants revealed changes in the activities of key enzymes mediating sucrose to starch conversion (ADP-glucose phosphorylase and sucrose synthase) and sucrose synthesis (sucrose phosphate synthase) leading to altered content of both soluble sugars and starch. Surprisingly the catecholamine level measured in transgenic plants was significantly increased; the reason for this is as yet unknown. However the presence of the receptor affected a broader range of enzyme activities than those affected by the massive accumulation of norepinephrine reported for plants over-expressing tyrosine decarboxylase. Therefore, it is suggested that the presence of the exogenous receptor activates catecholamine cAMP signalling in plants.ConclusionsOur data support the possible involvement of catecholamines in regulating plant carbon metabolism via cAMP signalling pathway.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry monitoring of anthocyanins in extracts from Arabidopsis thaliana leaves

Anthocyanins are secondary plant metabolites ubiquitous in the plant kingdom. They have different biological activities, so monitoring their content in plant tissue or in feed/food derived from plants may be an important task in different projects from various fields of molecular biology and biotechnology. Profiling of secondary metabolites with high-performance liquid chromatography/mass spectrometry (HPLC/MS) systems is time-consuming, especially when many samples have to be checked within a defined time frame with a reasonable number of repetitions according to the metabolomic standards. Even application of the advanced ultra-performance liquid chromatography (UPLC)/MS or equivalent systems would require a long time for analysis of numerous samples. We demonstrate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the assessment of level (concentration) of anthocyanins in leaf tissues of four Arabidopsis thaliana ecotypes grown at normal (20 degrees C/16 degrees C day/night) and decreased (4 degrees C) temperature. The quantitative results were obtained for anthocyanins with MALDI-TOF MS using ferulic acid as a matrix. The amounts of anthocyanins in leaves of A. thaliana varied from 0.3-2.5 microg per gram of leaves for ecotypes Col-0 and C24, respectively, and contents of these markedly increased in plants grown in the cold. The applied analytical method exhibited better repeatability of measurements than obtained with an HPLC/ion trap MS system.

Medicinal Bioprospecting of the Amazon Rainforest: A Modern Eldorado?

Ignorant of the New World, Europeans believed in El Dorado, a hidden city of immense wealth in gold. Many consider the Amazonian forest to be a medicinal treasure chest and potentially the largest drug dispensary in the world. Yet, the quest to obtain drugs from indigenous tropical plants remains elusive. Here, we assess the potential of new technologies to tap into the metabolic diversity of tropical plants. We also consider how regulations affect access to plant resources. We conclude that, although the road to this medicinal El Dorado may be long and arduous, many other smaller but still valuable finds are hidden along the way.

System-wide detection of protein-small molecule complexes suggests extensive metabolite regulation in plants

Protein small molecule interactions are at the core of cell regulation controlling metabolism and development. We reasoned that due to the lack of system wide approaches only a minority of those regulatory molecules are known. In order to see whether or not this assumption is true we developed an effective approach for the identification of small molecules having potential regulatory role that obviates the need of protein or small molecule baits. At the core of this approach is a simple biochemical co-fractionation taking advantage of size differences between proteins and small molecules. Metabolomics based analysis of small molecules co-fractionating with proteins identified a multitude of small molecules in Arabidopsis suggesting the existence of numerous, small molecules/metabolites bound to proteins representing potential regulatory molecules. The approach presented here uses Arabidopsis cell cultures, but is generic and hence applicable to all biological systems.

Affinity purification with metabolomic and proteomic analysis unravels diverse roles of nucleoside diphosphate kinases

We demonstrate that affinity purification is suited for analysing protein–small molecule interactions. Analysis of protein–protein–small molecule complexes of Arabidopsis nucleoside diphosphate kinases gave new insight into their function and regulation.

Interaction of 2',3'-cAMP with Rbp47b plays a role in stress granule formation

2′,3′-cAMP associates with Arabidopsis Rbp47b and plays a role in stress granule formation. 2′,3′-cAMP is an intriguing small molecule that is conserved among different kingdoms. 2′,3′-cAMP is presumably produced during RNA degradation, with increased cellular levels observed especially under stress conditions. Previously, we observed the presence of 2′,3′-cAMP in Arabidopsis (Arabidopsis thaliana) protein complexes isolated from native lysate, suggesting that 2′,3′-cAMP has potential protein partners in plants. Here, affinity purification experiments revealed that 2′,3′-cAMP associates with the stress granule (SG) proteome. SGs are aggregates composed of protein and mRNA, which enable cells to selectively store mRNA for use in response to stress such as heat whereby translation initiation is impaired. Using size-exclusion chromatography and affinity purification analyses, we identified Rbp47b, the key component of SGs, as a potential interacting partner of 2′,3′-cAMP. Furthermore, SG formation was promoted in 2′,3′-cAMP-treated Arabidopsis seedlings, and interactions between 2′,3′-cAMP and RNA-binding domains of Rbp47b, RRM2 and RRM3, were confirmed in vitro using microscale thermophoresis. Taken together, these results (1) describe novel small-molecule regulation of SG formation, (2) provide evidence for the biological role of 2′,3′-cAMP, and (3) demonstrate an original biochemical pipeline for the identification of protein-metabolite interactors.

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.