Fredrik Sterky

Gene Expression in Autumn Leaves

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula × tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.

A transcriptional timetable of autumn senescence

BackgroundWe have developed genomic tools to allow the genus Populus (aspens and cottonwoods) to be exploited as a full-featured model for investigating fundamental aspects of tree biology. We have undertaken large-scale expressed sequence tag (EST) sequencing programs and created Populus microarrays with significant gene coverage. One of the important aspects of plant biology that cannot be studied in annual plants is the gene activity involved in the induction of autumn leaf senescence.ResultsOn the basis of 36,354 Populus ESTs, obtained from seven cDNA libraries, we have created a DNA microarray consisting of 13,490 clones, spotted in duplicate. Of these clones, 12,376 (92%) were confirmed by resequencing and all sequences were annotated and functionally classified. Here we have used the microarray to study transcript abundance in leaves of a free-growing aspen tree (Populus tremula) in northern Sweden during natural autumn senescence. Of the 13,490 spotted clones, 3,792 represented genes with significant expression in all leaf samples from the seven studied dates.ConclusionsWe observed a major shift in gene expression, coinciding with massive chlorophyll degradation, that reflected a shift from photosynthetic competence to energy generation by mitochondrial respiration, oxidation of fatty acids and nutrient mobilization. Autumn senescence had much in common with senescence in annual plants; for example many proteases were induced. We also found evidence for increased transcriptional activity before the appearance of visible signs of senescence, presumably preparing the leaf for degradation of its components.

The genome sequence of black cottonwood (Populus trichocarpa) reveals 18 conserved cellulose synthase (CesA) genes

The genome sequence of Populus trichocarpa was screened for genes encoding cellulose synthases by using full-length cDNA sequences and ESTs previously identified in the tissue specific cDNA libraries of other poplars. The data obtained revealed 18 distinct CesA gene sequences in P. trichocarpa. The identified genes were grouped in seven gene pairs, one group of three sequences and one single gene. Evidence from gene expression studies of hybrid aspen suggests that both copies of at least one pair, CesA3-1 and CesA3-2, are actively transcribed. No sequences corresponding to the gene pair, CesA6-1 and CesA6-2, were found in Arabidopsis or hybrid aspen, while one homologous gene has been identified in the rice genome and an active transcript in Populus tremuloides. A phylogenetic analysis suggests that the CesA genes previously associated with secondary cell wall synthesis originate from a single ancestor gene and group in three distinct subgroups. The newly identified copies of CesA genes in P. trichocarpa give rise to a number of new questions concerning the mechanism of cellulose synthesis in trees.

MYB transcription factors are differentially expressed and regulated during secondary vascular tissue development in hybrid aspen

More than 120,000 poplar ESTs have been sequenced from 20 different cDNA libraries by the Swedish Centre for Tree Functional Genomics. We screened this EST collection for MYB transcription factors involved in secondary vascular tissue formation, and genes assigned as PttMYB3Ra, PttMYB4a and PttMYB21a were selected for further characterisation. Three MYB genes showed different expression patterns in various organs, tissues and stem sub-sections representing different developmental stages of vascular tissue formation. Furthermore, the analysis showed that PttMYB21a expression was much higher in secondary cell wall formation zone of xylem and phloem fibers than in other developmental zones. Transgenic hybrid aspen plants, expressing the 3′-part of the PttMYB21a gene in antisense orientation were generated to assess the function of PttMYB21a gene in vascular tissue formation and lignification. All transgenic lines showed reduced growth and had fewer internodes compared to the wild-type. The analysis of selected lines showed that acid soluble lignin present in the bark was higher in transgenic lines as compared to wild-type plants. Moreover a higher transcript level of caffeoyl-CoA 3-O-methyltransferase [CCoAOMT]; EC 2.1.1.104) was found in the phloem of the transgenic plants, suggesting that PttMYB21a might function as a transcriptional repressor.

Selection of protein epitopes for antibody production

Protein functional analysis in the post-genomic era is a huge task that has to be approached by different methods in parallel. The use of protein-specific antibodies in conjunction with tissue microarrays has proven to be one important technology. In this study, we present a strategy for the optimized design of protein subfragments for subsequent antibody production. The fragments are selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity. Furthermore, the fragments should have properties optimized for efficient protein production in Escherichia coli. The strategy has been implemented in Bishop, which is a Java-based software enabling the high-throughput production of protein fragments. Bishop allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides. A Basic Local Alignment Search Tool (BLAST) scanning procedure permits the selection of fragments of a selected size with a minimal sequence similarity to other proteins. The software and the strategy were evaluated on a human test data set and verified to fulfill the requested criteria.

Molecular cloning and characterization of a cDNA encoding poplar UDP-glucose dehydrogenase, a key gene of hemicellulose/pectin formation

Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of newly formed cell walls. A cDNA clone (Ugdh) corresponding to UGDH was isolated from a cDNA library prepared from cambial zone of poplar (Populus tremula x tremuloides). Within the 1824-nucleotide (nt)-long clone, an open reading frame encoded a protein of 481 amino acids (aa), with a calculated molecular weight of 53.1 kDa. The derived aa sequence showed 90% and 63% identity with UGDHs from soybean and bovine liver, respectively, and had highly conserved aa motifs believed to be of importance for nt binding and catalytic efficiency. In poplar, the Ugdh corresponds to one or two genes, as found by genomic Southern analysis. The gene was expressed predominantly in differentiating xylem and young leaves, with little expression in the phloem zone of the stem. The expression pattern matched that of UGDH protein, as found by immunoblotting. In leaves, the Ugdh expression was upregulated by a short-term feeding with sucrose, sorbitol and polyethylene glycol, and this effect was to some extent mimicked by light exposure. The data suggest that Ugdh is regulated via an osmoticum-dependent pathway, possibly related to the availability of osmotically active carbohydrate precursors to UDP-glucose, a substrate of UGDH.

Sequence analysis of genes and genomes

A major step towards understanding of the genetic basis of an organism is the complete sequence determination of all genes in its genome. The development of powerful techniques for DNA sequencing has enabled sequencing of large amounts of gene fragments and even complete genomes. Important new techniques for physical mapping, DNA sequencing and sequence analysis have been developed. To increase the throughput, automated procedures for sample preparation and new software for sequence analysis have been applied. This review describes the development of new sequencing methods and the optimisation of sequencing strategies for whole genome and cDNA analysis, as well as discusses issues regarding sequence analysis and annotation.

Chromosomal sequencing using a PCR-based biotin-capture method allowed isolation of the complete gene for the outer membrane protein A of Klebsiella pneumoniae.

By employing a novel biotin- and PCR-assisted capture method, which allows determination of unknown sequences on chromosomal DNA, the gene for the outer membrane protein A (OmpA) of Klebsiella pneumoniae has been isolated and sequenced to completion. The method involves linear amplification of DNA from a biotinylated primer annealing to a region with known sequence. After capture of the amplified single-stranded DNA on to paramagnetic beads, unspecifically annealing primers, i.e. arbitrary primers, were used to generate sequences with only partly determined nt sequences. The homology of the sequenced gene to ompA of related bacteria is discussed, and the gene fragment was assembled for intracellular expression in Escherichia coli, and two different fusion proteins were produced and recovered with good yields. The importance of the novel chromosomal sequencing method for gene isolation in general and the potential use of the OmpA fusion proteins are discussed.

The sequence of a 30 kb fragment on the left arm of chromosome XV from Saccharomyces cerevisiae reveals 15 open reading frames, five of which correspond to previously identified genes

We report the sequence of a 30 469 bp long DNA fragment on the left arm of chromosome XV of Saccharomyces cerevisiae. The fragment contains 15 open reading frames (ORFs) of at least 300 bp. Five previously sequenced yeast genes, PHO80, TIR2, SLG1, the gene encoding the subtilisin‐like protease III precursor and the gene coding for ATP‐dependent permease, are found among these ORFs. By DNA sequence comparison, two ORFs identified previously reported expressed sequence tags from yeast. Of the proteins encoded by the remaining eight ORFs, six show similarities to proteins from different organisms and two lack detectable similarity with any amino acid sequence described in public data banks. The DNA sequence has been deposited in GenBank under Accession Number U43491.

Analysis of 70,000 EST sequences to study divergence between two closely related Populus species

The Populus genus has evolved as the model organism for forest tree genomics, which has been further emphasised with the sequencing of the Populus trichocarpa genome. Populus species are widely spread over the Northern Hemisphere and provide a great source of genetic diversity, which can be used for mapping of quantitative trait loci, positional cloning, association mapping and studies in environmental adaptation. Collections of expressed sequence tags (ESTs) are rich sources in studies of genetic diversity. Here, we report on an in-depth analysis of 70,000 ESTs from two Populus species, Populus tremula and Populus trichocarpa. We present data on the level of conservation in transcript sequences and supply a collection of potential single nucleotide polymorphisms.