Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

Gene expression profiling of single epidermal, basal and trichome cells of Arabidopsis thaliana.

Samples of single epidermal, basal and trichome cells were collected by glass microcapillaries from 7-week-old Arabidopsis thaliana leaves. Transcript amplification of these single-cell samples was performed by RT PCR. For gene expression profiling, we hybridized the amplified transcriptome of each individual cell type to nylon membranes spotted with 16,000 Arabidopsis expressed sequence tags (ESTs). Initial analysis of the array filter data enabled us to functionally categorize transcripts that were present in each individual cell type. In order to confirm the filter array data, we used RT PCR. Results of this RT PCR approach confirmed the presence of 12 selected candidate genes in agreement with array filter hybridization data. Further, transcripts involved in detoxification and sulfur metabolism could be identified in epidermal cell extracts. Together, the results of our study provide the localization of approximately 1000 expressed genes to either pavement, basal or trichome cells. To cluster transcripts with similar expression levels, we developed a novel mathematical algorithm. Based on the mean and standard deviation, ratios of expression levels of a transcript were defined for pairs of the three cell types. This numerical analysis enabled subdivision into 67 categories of genes differentially expressed in epidermal, basal and trichome cells. Transcripts in each category displayed similar ratios of expression levels in the three cell types. Examples of these clusters are presented and discussed in Appendix A.

Delineation of species within the Trichoderma viride/atroviride/koningii complex by UP-PCR cross-blot hybridization.

Sequences from the ribosomal DNA ITS regions have shown that there is limited variation among the species in the Trichoderma viride/atroviride/koningii species complex also known as Hypocrea rufa complex, and that infraspecies variation sometimes is larger than interspecies variation. Strains belonging to T. viride, T. atroviride, T. koningii, T. asperellum, and respective teleomorphs were analyzed using cross-blot hybridization of PCR products generated by the universally primed PCR (UP-PCR) technique. The hybridization results showed that the morphologically defined species could be delineated molecularly, suggesting that the species are more separated than indicated by ITS sequence phylogeny. However, cross-hybridization signals at infra- and interspecies level within this species complex was overlapping, again raising the question of how many species there are. Due to the heterogeneity of T. viride revealed, a further revision of this species is needed. In addition, this study shows that a macroarray (DNA chip) containing membrane-bound UP-PCR products for reference strains can be developed for routine identification of Trichoderma strains.

Hypocrea atroviridis sp. nov., the teleomorph of Trichoderma atroviride.

A new species, Hypocrea atroviridis, is described for the teleomorph of Trichoderma atroviride. Based on sequences of ITS-1, 5.8S, and ITS-2 regions of the rDNA complex and translation-elongation factor (EF-1α), T. atroviride and H. atroviridis form a well-supported clade within Trichoderma sect. Trichoderma. The conserved anamorphic phenotype of T. atroviride, observed for both conidial and ascospore derived cultures, was only found within that clade. In contrast, the teleomorph phenotype of H. atroviridis was morphologically indistinguishable from H. rufa, the teleomorph of T. viride. This Hypocrea phenotype may, therefore, be considered to be plesiomorphic within Trichoderma sect. Trichoderma, suggesting that genes controlling the expression of the teleomorph and anamorph evolve at different rates and that the genes controlling expression of the teleomorph are more conserved than are those controlling the expression of the anamorph.