Bart Panis

PROTEOME ANALYSIS OF NON-MODEL PLANTS: A CHALLENGING BUT POWERFUL APPROACH

Biological research has focused in the past on model organisms and most of the functional genomics studies in the field of plant sciences are still performed on model species or species that are characterized to a great extent. However, numerous non-model plants are essential as food, feed, or energy resource. Some features and processes are unique to these plant species or families and cannot be approached via a model plant. The power of all proteomic and transcriptomic methods, that is, high-throughput identification of candidate gene products, tends to be lost in non-model species due to the lack of genomic information or due to the sequence divergence to a related model organism. Nevertheless, a proteomics approach has a great potential to study non-model species. This work reviews non-model plants from a proteomic angle and provides an outline of the problems encountered when initiating the proteome analysis of a non-model organism. The review tackles problems associated with (i) sample preparation, (ii) the analysis and interpretation of a complex data set, (iii) the protein identification via MS, and (iv) data management and integration. We will illustrate the power of 2DE for non-model plants in combination with multivariate data analysis and MS/MS identification and will evaluate possible alternatives.

Genetic Transformation of Banana and Plantain (Musa spp.) via Particle Bombardment

We have developed a simple protocol to allow the production of transgenic banana plants. Foreign genes were delivered into embryogenic suspension cells using accelerated particles coated with DNA. Bombardment parameters were optimized for a modified particle gun resulting in high levels of transient expression of the β-glucuronidase gene in both banana and plantain cells. Bombarded banana cells were selected with hygromycin and regenerated into plants. Molecular and histochemical characterization of transformants revealed the stable integration of the transferred genes into the banana genome.

Cryopreservation for the elimination of cucumber mosaic and banana streak viruses from banana (Musa spp.).

Abstract. The utilisation of cryopreservation for the eradication of cucumber mosaic virus (CMV) or banana streak virus (BSV) from Musa spp. was investigated. Banana plants, cv. Williams (AAA, Cavendish subgroup), were mechanically infected with CMV or naturally infected with BSV, and proliferating meristems were produced from the infected plants. Excised meristematic clumps were cryopreserved through vitrification using PVS-2 solution. The health status of regenerated in vitro plants was first checked by means of ELISA. The putative virus-free material was subsequently tested a second time following greenhouse acclimatisation. The frequency of virus eradication for CMV and BSV was 30% and 90%, respectively, following cryopreservation. In comparison, the frequency of virus-free plants regenerated directly from highly proliferating meristems, corresponding to a spontaneous eradication rate, reached 0% and 52% for CMV and BSV, respectively. The conventional meristem culture resulted in 0% CMV-free plants and 76% BSV-free plants, while the cryoprotective treatment resulted in 2% CMV-free plants and 87% BSV-free plants. To understand the mode of action of cryopreservation for the eradication of viral particles, we examined the structure of the meristem tips by light microscopy. The cryopreservation method used only allowed survival of small areas of cells located in the meristematic dome and at the base of the primordia.

Ultrastructural changes associated with cryopreservation of banana ( Musa spp.) highly proliferating meristems.

Abstract.Cryopreservation has been shown to improve the frequency of virus elimination – specifically cucumber mosaic virus and banana streak virus – from banana (Musa spp.) plants. To understand the mode of action of cryopreservation for the eradication of viral particles, we examined the ultrastructure of meristem tips at each step of the cryopreservation process. Excised meristematic clumps produced from infected banana plants belonging to cv. Williams (AAA, Cavendish subgroup) were cryopreserved through vitrification using the PVS-2 solution. We demonstrated that the cryopreservation method used only allowed survival of small areas of cells in the meristematic dome and at the base of the primordia. Cellular and subcellular changes occurring during the cryopreservation process are discussed.

Treatment of missing values for multivariate statistical analysis of gel‐based proteomics data

The presence of missing values in gel‐based proteomics data represents a real challenge if an objective statistical analysis is pursued. Different methods to handle missing values were evaluated and their influence is discussed on the selection of important proteins through multivariate techniques. The evaluated methods consisted of directly dealing with them during the multivariate analysis with the nonlinear estimation by iterative partial least squares (NIPALS) algorithm or imputing them by using either k‐nearest neighbor or Bayesian principal component analysis (BPCA) before carrying out the multivariate analysis. These techniques were applied to data obtained from gels stained with classical postrunning dyes and from DIGE gels. Before applying the multivariate techniques, the normality and homoscedasticity assumptions on which parametric tests are based on were tested in order to perform a sound statistical analysis. From the three tested methods to handle missing values in our datasets, BPCA imputation of missing values showed to be the most consistent method.

Functional genomics in a non-model crop: transcriptomics or proteomics?

There is no question that protein- and RNA-based measurements are complementary, but which approach has the highest return in the case of a non-model crop and what is the correlation between mRNA and proteins? We describe and evaluate in detail the advantages and pitfalls of both a proteomics and a transcriptomics approach. The information on the abundance of transcripts was obtained by serial analysis of gene expression (SAGE), while information on the abundance of proteins was obtained via two-dimensional gel electrophoresis.

Screening the banana biodiversity for drought tolerance: can an in vitro growth model and proteomics be used as a tool to discover tolerant varieties and understand homeostasis

There is a great need for research aimed at understanding drought tolerance, screening for drought tolerant varieties and breeding crops with an improved water use efficiency. Bananas and plantains are a major staple food and export product with a worldwide production of over 135 million tonnes per year. Water however is the most limiting abiotic factor in banana production. A screening of the Musa biodiversity has not yet been performed. We at KU Leuven host the Musa International Germplasm collection with over 1200 accessions. To screen the Musa biodiversity for drought tolerant varieties, we developed a screening test for in vitro plants. Five varieties representing different genomic constitutions in banana (AAAh, AAA, AAB, AABp, and ABB) were selected and subjected to a mild osmotic stress. The ABB variety showed the smallest stress induced growth reduction. To get an insight into the acclimation and the accomplishment of homeostasis, the leaf proteome of this variety was characterized via 2D DIGE. After extraction of the leaf proteome of six control and six stressed plants, 2600 spots could be distinguished. A PCA analysis indicates that control and stressed plants can blindly be classified based on their proteome. One hundred and twelve proteins were significantly more abundant in the stressed plants and 18 proteins were significantly more abundant in control plants (FDR α 0.05). Twenty four differential proteins could be identified. The proteome analysis clearly shows that there is a new balance in the stressed plants and that the respiration, metabolism of ROS and several dehydrogenases involved in NAD/NADH homeostasis play an important role.

Arbuscular mycorrhizal fungi reduce root-knot nematode penetration through altered root exudation of their host

AimsArbuscular mycorrhizal fungi (AMF) can control root-knot nematode infection, but the mode of action is still unknown. We investigated the effects of AMF and mycorrhizal root exudates on the initial steps of Meloidogyne incognita infection, namely movement towards and penetration of tomato roots.MethodsM. incognita soil migration and root penetration were evaluated in a twin-chamber set-up consisting of a control and mycorrhizal (Glomus mosseae) plant compartment (Solanum lycopersicum cv. Marmande) connected by a bridge. Penetration into control and mycorrhizal roots was also assessed when non-mycorrhizal or mycorrhizal root exudates were applied and nematode motility in the presence of the root exudates was tested in vitro.ResultsM. incognita penetration was significantly reduced in mycorrhizal roots compared to control roots. In the twin-chamber set-up, equal numbers of nematodes moved to both compartments, but the majority accumulated in the soil of the mycorrhizal plant compartment, while for the control plants the majority penetrated the roots. Application of mycorrhizal root exudates further reduced nematode penetration in mycorrhizal plants and temporarily paralyzed nematodes, compared with application of water or non-mycorrhizal root exudates.ConclusionsNematode penetration was reduced in mycorrhizal tomato roots and mycorrhizal root exudates probably contributed at least partially by affecting nematode motility.

Structure and regulation of the Asr gene family in banana

Abscisic acid, stress, ripening proteins (ASR) are a family of plant-specific small hydrophilic proteins. Studies in various plant species have highlighted their role in increased resistance to abiotic stress, including drought, but their specific function remains unknown. As a first step toward their potential use in crop improvement, we investigated the structure and regulation of the Asr gene family in Musa species (bananas and plantains). We determined that the MusaAsr gene family contained at least four members, all of which exhibited the typical two exons, one intron structure of Asr genes and the “ABA/WDS” (abscisic acid/water deficit stress) domain characteristic of Asr genes. Phylogenetic analyses determined that the MusaAsr genes were closely related to each other, probably as the product of recent duplication events. For two of the four members, two versions corresponding to the two sub-genomes of Musa, acuminata and balbisiana were identified. Gene expression and protein analyses were performed and Asr expression could be detected in meristem cultures, root, pseudostem, leaf and cormus. In meristem cultures, mAsr1 and mAsr3 were induced by osmotic stress and wounding, while mAsr3 and mAsr4 were induced by exposure to ABA. mASR3 exhibited the most variation both in terms of amino acid sequence and expression pattern, making it the most promising candidate for further functional study and use in crop improvement.

A workflow for peptide-based proteomics in a poorly sequenced plant: A case study on the plasma membrane proteome of banana

Membrane proteins are an interesting class of proteins because of their functional importance. Unfortunately their analysis is hampered by low abundance and poor solubility in aqueous media. Since shotgun methods are high-throughput and partly overcome these problems, they are preferred for membrane proteomics. However, their application in non-model plants demands special precautions to prevent false positive identification of proteins. In the current paper, a workflow for membrane proteomics in banana, a poorly sequenced plant, is proposed. The main steps of this workflow are (i) optimization of the peptide separation, (ii) performing de novo sequencing to allow a sequence homology search and (iii) visualization of identified peptide-protein associations using Cytoscape to remove redundancy and wrongly assigned peptides, based on species-specific information. By applying this workflow, integral plasma membrane proteins from banana leaves were successfully identified.