Erik Alexandersson

Whole gene family expression and drought stress regulation of aquaporins

Since many aquaporins (AQPs) act as water channels, they are thought to play an important role in plant water relations. It is therefore of interest to study the expression patterns of AQP isoforms in order to further elucidate their involvement in plant water transport. We have monitored the expression patterns of all 35 Arabidopsis AQPs in leaves, roots and flowers by cDNA microarrays, specially designed for AQPs, and by quantitative real-time reverse transcriptase PCR (Q-RT-PCR). This showed that many AQPs are pre-dominantly expressed in either root or flower organs, whereas no AQP isoform seem to be leaf specific. Looking at the AQP subfamilies, most plasma membrane intrinsic proteins (PIPs) and some tonoplast intrinsic proteins (TIPs) have a high level of expression, while NOD26-like proteins (NIPs) are present at a much lower level. In addition, we show that PIP transcripts are generally down-regulated upon gradual drought stress in leaves, with the exception of AtPIP1;4 and AtPIP2;5, which are up-regulated. AtPIP2;6 and AtSIP1;1 are constitutively expressed and not significantly affected by the drought stress. The transcriptional down-regulation of PIP genes upon drought stress could also be observed on the protein level.

Transcriptional regulation of aquaporins in accessions of Arabidopsis in response to drought stress.

Aquaporins facilitate water transport over cellular membranes, and are therefore believed to play an important role in water homeostasis. In higher plants aquaporin-like proteins, also called major intrinsic proteins (MIPs), are divided into five subfamilies. We have previously shown that MIP transcription in Arabidopsis thaliana is generally downregulated in leaves upon drought stress, apart from two members of the plasma membrane intrinsic protein (PIP) subfamily, AtPIP1;4 and AtPIP2;5, which are upregulated. In order to assess whether this regulation is general or accession-specific we monitored the gene expression of all PIPs in five Arabidopsis accessions. The overall drought regulation of PIPs was well conserved for all five accessions tested, suggesting a general and fundamental physiological role of this drought response. In addition, significant differences among accessions were identified for transcripts of three PIP genes. Principal component analysis showed that most of the PIP transcriptional variation during drought stress could be explained by one variable linked to leaf water content. Promoter-GUS constructs of AtPIP1;4, AtPIP2;5 and also AtPIP2;6, which is unresponsive to drought stress, had distinct expression patterns concentrated in the base of the leaf petioles and parts of the flowers. The presence of drought stress response elements within the 1.6-kb promoter regions of AtPIP1;4 and AtPIP2;5 was demonstrated by comparing transcription of the promoter reporter construct and the endogenous gene upon drought stress. Analysis by ATTED-II and other web-based bioinformatical tools showed that several of the MIPs downregulated upon drought are strongly co-expressed, whereas AtPIP1;4, AtPIP2;5 and AtPIP2;6 are not co-expressed.

The genes and enzymes of the carotenoid metabolic pathway in Vitis vinifera L.

BackgroundCarotenoids are a heterogeneous group of plant isoprenoids primarily involved in photosynthesis. In plants the cleavage of carotenoids leads to the formation of the phytohormones abscisic acid and strigolactone, and C13-norisoprenoids involved in the characteristic flavour and aroma compounds in flowers and fruits and are of specific importance in the varietal character of grapes and wine. This work extends the previous reports of carotenoid gene expression and photosynthetic pigment analysis by providing an up-to-date pathway analysis and an important framework for the analysis of carotenoid metabolic pathways in grapevine.ResultsComparative genomics was used to identify 42 genes putatively involved in carotenoid biosynthesis/catabolism in grapevine. The genes are distributed on 16 of the 19 chromosomes and have been localised to the physical map of the heterozygous ENTAV115 grapevine sequence. Nine of the genes occur as single copies whereas the rest of the carotenoid metabolic genes have more than one paralogue. The cDNA copies of eleven corresponding genes from Vitis vinifera L. cv. Pinotage were characterised, and four where shown to be functional. Microarrays provided expression profiles of 39 accessions in the metabolic pathway during three berry developmental stages in Sauvignon blanc, whereas an optimised HPLC analysis provided the concentrations of individual carotenoids. This provides evidence of the functioning of the lutein epoxide cycle and the respective genes in grapevine. Similarly, orthologues of genes leading to the formation of strigolactone involved in shoot branching inhibition were identified: CCD7, CCD8 and MAX1. Moreover, the isoforms typically have different expression patterns, confirming the complex regulation of the pathway. Of particular interest is the expression pattern of the three VvNCEDs: Our results support previous findings that VvNCED3 is likely the isoform linked to ABA content in berries.ConclusionsThe carotenoid metabolic pathway is well characterised, and the genes and enzymes have been studied in a number of plants. The study of the 42 carotenoid pathway genes of grapevine showed that they share a high degree of similarity with other eudicots. Expression and pigment profiling of developing berries provided insights into the most complete grapevine carotenoid pathway representation. This study represents an important reference study for further characterisation of carotenoid biosynthesis and catabolism in grapevine.

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

BackgroundWhen performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast Pichia pastoris.ResultsBy comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.ConclusionsWe report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant P. pastoris clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of P. pastoris clones with high expression levels of aquaporins and other classes of membrane proteins.

Optimisation of an HPLC method for the simultaneous quantification of the major sugars and organic acids in grapevine berries

A high performance liquid chromatographic method was developed to profile major sugars and organic acids in grapevine berries. Sugars and organic acids in grapevine berries were extracted by chloroform/polyvinylpolypyrrolidone purification. The extracts were chromatographed on an Aminex HPX-87H ion-exchange HPLC column with 5mM sulphuric acid as mobile phase. Chromatography was visualised via a diode array detector combined with a refractive index detector. The analysis was calibrated using external standard calibration and a novel equation was used to calculate the concentrations of malic acid and fructose from unresolved separation. For the method to be utilised for analysing a large numbers of berry samples, each sample was directly injected after sample extraction and the extraction step was downscaled to allow the use of small amounts of sample material. The concentrations of sugars and organic acids in grapevine berry samples were normalised to the internal standard concentrations obtained after extraction of an internal standard mixture. The analysis method exhibits a good precision and a high analyte recovery from samples spiked with the standard mixture and is suitable for the profiling of major sugars and organic acids in grapevine berry samples at different stages of berry development. This is the first report on the combined profiling of the major sugars and organic acids in grapevine berries using milligram amounts of plant material with direct injection after sample extraction.

The effects of the loss of TIP1;1 and TIP1;2 aquaporins in Arabidopsis thaliana

Loss of aquaporin TIP1;1 in Arabidopsis has been suggested to result in early senescence and plant death. This was based on the fact that a partial reduction of TIP1;1 by RNA interference (RNAi) led to gradual phenotypes, ranging from indistinguishable from wild type to lethality, depending on the degree of downregulation of the target messenger, and displaying pleiotropic effects in primary metabolism and cell signalling. A hypothesis was put forward to suggest that TIP1;1, apart from its transport function, may play an essential role in vesicle routing. Here we identify an Arabidopsis transposon insertion line tip1;1-1 that is completely devoid of TIP1;1 protein, as demonstrated by western blotting and immunolocalization using an isoform-specific antibody. Strikingly, the complete absence of the protein did not result in any significant effect on metabolism or elemental composition of the plants. Microarray analysis did not indicate increased expression of other aquaporins to compensate for the lack of TIP1;1 in tip1;1-1. We further developed a double mutant of TIPs in Arabidopsis, lacking both TIP1;1 and its closest paralog TIP1;2. Arabidopsis mutants lacking both TIP1;1 and TIP1;2 showed a minor increase in anthocyanin content, and a reduction in catalase activity, but showed no changes in water status. In contrast to earlier reports, plants lacking TIP1;1 and TIP1;2 aquaporins are alive and thriving. We suggest that RNAi directed towards TIP1;1 may have resulted in off-target gene silencing, a notion that is potentially interesting for various studies analysing gene function by RNAi.

Plant secretome proteomics

The plant secretome refers to the set of proteins secreted out of the plant cell into the surrounding extracellular space commonly referred to as the apoplast. Secreted proteins maintain cell structure and acts in signaling and are crucial for stress responses where they can interact with pathogen effectors and control the extracellular environment. Typically, secreted proteins contain an N-terminal signal peptide and are directed through the endoplasmic reticulum/Golgi pathway. However, in plants many proteins found in the secretome lack such a signature and might follow alternative ways of secretion. This review covers techniques to isolate plant secretomes and how to identify and quantify their constituent proteins. Furthermore, bioinformatical tools to predict secretion signals and define the putative secretome are presented. Findings from proteomic studies and important protein families of plant secretomes, such as proteases and hydrolases, are highlighted.

Normalyzer: A Tool for Rapid Evaluation of Normalization Methods for Omics Data Sets

High-throughput omics data often contain systematic biases introduced during various steps of sample processing and data generation. As the source of these biases is usually unknown, it is difficult to select an optimal normalization method for a given data set. To facilitate this process, we introduce the open-source tool “Normalyzer”. It normalizes the data with 12 different normalization methods and generates a report with several quantitative and qualitative plots for comparative evaluation of different methods. The usefulness of Normalyzer is demonstrated with three different case studies from quantitative proteomics and transcriptomics. The results from these case studies show that the choice of normalization method strongly influences the outcome of downstream quantitative comparisons. Normalyzer is an R package and can be used locally or through the online implementation at http://quantitativeproteomics.org/normalyzer.

Phosphite-induced changes of the transcriptome and secretome in Solanum tuberosum leading to resistance against Phytophthora infestans

BackgroundPotato late blight caused by the oomycete pathogen Phytophthora infestans can lead to immense yield loss. We investigated the transcriptome of Solanum tubersoum (cv. Desiree) and characterized the secretome by quantitative proteomics after foliar application of the protective agent phosphite. We also studied the distribution of phosphite in planta after application and tested transgenic potato lines with impaired in salicylic and jasmonic acid signaling.ResultsPhosphite had a rapid and transient effect on the transcriptome, with a clear response 3 h after treatment. Strikingly this effect lasted less than 24 h, whereas protection was observed throughout all time points tested. In contrast, 67 secretome proteins predominantly associated with cell-wall processes and defense changed in abundance at 48 h after treatment. Transcripts associated with defense, wounding, and oxidative stress constituted the core of the phosphite response. We also observed changes in primary metabolism and cell wall-related processes. These changes were shown not to be due to phosphate depletion or acidification caused by phosphite treatment. Of the phosphite-regulated transcripts 40% also changed with β-aminobutyric acid (BABA) as an elicitor, while the defence gene PR1 was only up-regulated by BABA. Although phosphite was shown to be distributed in planta to parts not directly exposed to phosphite, no protection in leaves without direct foliar application was observed. Furthermore, the analysis of transgenic potato lines indicated that the phosphite-mediated resistance was independent of the plant hormones salicylic and jasmonic acid.ConclusionsOur study suggests that a rapid phosphite-triggered response is important to confer long-lasting resistance against P. infestans and gives molecular understanding of its successful field applications.

Constitutive expression of a grapevine polygalacturonase-inhibiting protein affects gene expression and cell wall properties in uninfected tobacco

BackgroundPolygalacturonase-inhibiting proteins (PGIPs) directly limit the effective ingress of fungal pathogens by inhibiting cell wall-degrading endopolygalacturonases (ePGs). Transgenic tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1 have previously been shown to be resistant to Botrytis infection. In this study we characterized two of these PGIP over-expressing lines with known resistance phenotypes by gene expression and hormone profiling in the absence of pathogen infection.ResultsGlobal gene expression was performed by a cross-species microarray approach using a potato cDNA microarray. The degree of potential cross-hybridization between probes was modeled by a novel computational workflow designed in-house. Probe annotations were updated by predicting probe-to-transcript hybridizations and combining information derived from other plant species. Comparing uninfected Vvpgip1-overexpressing lines to wild-type (WT), 318 probes showed significant change in expression. Functional groups of genes involved in metabolism and associated to the cell wall were identified and consequent cell wall analysis revealed increased lignin-levels in the transgenic lines, but no major differences in cell wall-derived polysaccharides. GO enrichment analysis also identified genes responsive to auxin, which was supported by elevated indole-acetic acid (IAA) levels in the transgenic lines. Finally, a down-regulation of xyloglucan endotransglycosylase/hydrolases (XTHs), which are important in cell wall remodeling, was linked to a decrease in total XTH activity.ConclusionsThis evaluation of PGIP over-expressing plants performed under pathogen-free conditions to exclude the classical PGIP-ePG inhibition interaction indicates additional roles for PGIPs beyond the inhibition of ePGs.