Biomedical subjects

Julia Kehr

Publications and source records attributed to Julia Kehr.

6 recordsLinked to original sources

PubMed2004

Xylem sap protein composition is conserved among different plant species.

Xylem sap from broccoli (Brassica oleracea L. cv. Calabrais), rape (Brassica napus L. cv. Drakkar), pumpkin (Cucurbita maxima Duch. cv. gelber Zentner) and cucumber (Cucumis sativus L. cv. Hoffmanns Giganta) was collected by root pressure exudation from the surface of cut stems of healthy, adult plants. Total protein concentrations were in the range of 100 microg ml(-1). One-dimensional gel electrophoresis (SDS-PAGE) resulted in 10-20 visible protein bands in a molecular mass range from 10 to 100 kDa. The main bands were cut out, digested with trypsin, and analysed using tandem mass spectrometry. Fifty bands resulted in amino acid sequence information that was used to perform database similarity searches. Sequences from 30 bands showed high homology to proteins present in databases. Among them, we found mostly peroxidases, but could also identify the lectin-like xylem protein XSP30, a glycine-rich protein, serine proteases, an aspartyl protease family protein, chitinases, and a lipid transfer protein-like polypeptide. Sequence analysis predicted apoplastic secretion signals for all database entries similar to the partial xylem protein sequences. This and the lack of cross-reactivity with phloem protein-specific antibodies suggest that the proteins really originate from the xylem and do not result from phloem contamination. Most of the highly similar proteins probably function in repair and defence reactions. Some of the most abundant proteins (peroxidases, chitinases, serine proteases) were present in xylem exudate of all species analysed, often in more than one band. This indicates an important basic role of these proteins in maintaining xylem function.

Amino Acid Sequence↗

PubMed2004

Proteomics of curcurbit phloem exudate reveals a network of defence proteins.

Many different proteins can be separated from the sap of mature sieve tubes of different plant species. To date, only a limited number of those have been identified and functionally characterised. Due to sieve tubes inability of transcription and translation, the proteins are most probably synthesised in the intimately connected companion cells and transported into the sieve elements through plasmodesmata. The specific protein composition of phloem sap suggests an important role of these proteins not only for sieve tube maintenance, but also for whole plant physiology and development. Here we describe a comprehensive analysis of the phloem protein composition employing one- and high-resolution two-dimensional gel electrophoresis and partial sequencing by mass spectrometry. In this study more than 300 partial sequences generated by hybrid mass spectrometry were used to identify a total of 45 different proteins from the phloem exudates of cucumber (Cucumis sativus L. cv. Hoffmanns Giganta) and pumpkin (Cucurbita maxima Duch. cv. Gelber Zentner) plants. In addition to previously described phloem proteins, it was possible to localise proteins with high similarity to an acyl-CoA binding protein, a glyoxalase, a malate dehydrogenase, a rhodanese-like protein, a drought-induced protein, and a beta-glucosidase. The results indicate that the majority of the so far identified proteins are involved in stress and defence reactions.

Cucumis sativus↗

PubMed2003

Overexpression of the sucrose transporter SoSUT1 in potato results in alterations in leaf carbon partitioning and in tuber metabolism but has little impact on tuber morphology.

The aim of this work was to examine the consequences of the heterologous expression of a spinach ( Spinacia oleracea L.) sucrose transporter ( SoSUT1) in potato ( Solanum tuberosum L.). Many studies have indicated that reduction of the expression of this class of sucrose transporter has deleterious effects on plant growth and development; however, until now the possibility of improving plant performance by enhancing the expression of this sucrose transporter has not been reported. With this intention we constructed a chimeric construct in which SoSUT1 was cloned in-frame with the myc epitope. We confirmed that this construct, SoSUT1m, was able to mediate sucrose transport by expression in the yeast strain SUSY7. SoSUT1m was expressed in wild-type potato in the sense orientation under the control of the cauliflower mosaic virus 35S promoter to evaluate the effect of an increased constitutive expression of a class-I sucrose transporter. We confirmed that these plants displayed expression of SoSUT1 at both the transcript and protein level and that microsomal fragments isolated from selected lines had an increased sucrose uptake capacity. Analysis of metabolism of these lines indicated that the leaves were characterised by a reduced sucrose level yet exhibited little change in photosynthetic rate. Furthermore, despite the observed increase in sugar (and reduction in amino acid) levels within the tubers, there was little change in either starch content or tuber yield in the transformants. In summary, the genetic manipulation described in this paper resulted in a shift in carbon partitioning in both leaves and tubers and an increased sucrose uptake rate in plasma-membrane vesicles isolated from these lines, but had little impact on tuber metabolism or morphology.

Carbohydrate Metabolism↗

PubMed2003

Single cell technology.

Complexity is a fundamental feature of life. Like animals, higher plants consist of a multitude of different distinct tissues and cell types, each contributing to the overall performance of the whole organism. Our understanding and knowledge of physiology will greatly increase as our ability to spatially resolve molecular and biochemical processes improves. Differential analysis of individual tissues and single cells will eliminate the averaging effect and allow the discovery of detailed differences between various cell types. Recent breakthroughs have been made in tissue-specific DNA, RNA and protein analysis of plants by applying laser-based microdissection techniques.

DNA, Plant↗

PubMed2002

Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes.

The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.

Amino Acid Sequence↗

PubMed2002

Using array hybridization to monitor gene expression at the single cell level.

Advances in high-throughput genome sequencing demand the development of more efficient ways of examining gene expression at a cellular level. During recent years, polymerase chain reaction (PCR)-based methods have been developed that allow the amplification of mRNA from small amounts of material, even from single animal cells. In parallel, several analytical tools permit a global monitoring of gene expression. To date, high throughput analysis methods have not been accessible for single plant cell samples. In the protocol described here, cDNA array hybridization (expression profiling) and an amplification strategy using reverse transcriptase PCR are merged with high spatial resolution sampling from undamaged plant tissue. This protocol gives us a new tool to examine tissue-specific gene expression patterns on a comprehensive scale. To demonstrate the usefulness of this tool, gene expression patterns in samples from Arabidopsis thaliana L. cv. C24 leaf epidermal and mesophyll cells were measured; several differentially expressed genes were identified when single cell samples were compared. The protocol described has the potential of increasing the efficiency of tissue-specific expression analysis by combining high-throughput profiling with straightforward sampling and amplification procedures.

Arabidopsis↗