An Arabidopsis mutant disrupted in valine catabolism is also compromised in peroxisomal fatty acid β-oxidation

AbstractCharacterisation of the Arabidopsis dbr5 mutant, which was isolated on the basis of 2,4-dichlorophenoxybutyric acid (2,4-DB) resistance, revealed that it is disrupted in the CHY1 gene. CHY1 encodes a peroxisomal protein that is 43% identical to the mammalian β-hydroxyisobutryl-CoA hydrolase of valine catabolism. We show that 2,4-DB resistance and the associated sucrose dependent seedling growth are due to a large activity decrease of 3-ketoacyl-CoA thiolase, which is involved in peroxisomal fatty acid β-oxidation. 14C-feeding studies demonstrate that dbr5 and chy1 seedlings are reduced in valine catabolism. These data support the hypothesis that CHY1 plays a key role in peroxisomal valine catabolism and that disruption of this enzyme results in accumulation of a toxic intermediate, methacrylyl-CoA, that inhibits 3-ketoacyl-CoA thiolase activity and thus blocks peroxisomal β-oxidation. We also show that CHY1 is repressed in seedlings grown on sugars, which suggests that branched chain amino acid catabolism is transcriptionally regulated by nutritional status

Tools for nuclear transformation in Chlamydomonas reinhardtii

Due to its simple life cycle and classical Mendelian genetics the green alga Chlamydomonas reinhardtii has long been used as a model system for the study of flagellar function, photosynthesis and response to light. Transformation of the nuclear genome has now become routine and with this the development of a variety of promoters, transformation markers and methods for genomic complementation and insertional mutagenesis has taken place. However, in order to exploit fully the potential of Chlamydomonas, further procedures for molecular genetic investigation are required. For example, it is desirable to develop a detailed molecular map of the Chlamydomonas nuclear genome. Also there is a need to find a suitable reporter molecule that can act as a marker for gene expression and protein localisation. Other desirable advances would include a way of targeting specific genes by selection for rare homologous recombination events and a means for down- regulation of genes by the application of antisense technology. This thesis presents the results of investigation into possible new tools for nuclear transformation. Firstly, the results of a pilot study of analysis of Expressed Sequence Tags from Chlamydomonas are presented. Sixteen cDNAs chosen at random from a Chlamydomonas cDNA library were sequenced. The sequences were analysed for sequence similarities to other organisms and for functional motifs. Secondly, the use of Green Fluorescent Protein as a reporter molecule for Chlamydomonas was investigated. The GFP coding sequence under control of a Chlamydomonas promoter was introduced into the nuclear genome and the transformants examined for expression of Green Fluorescent Protein. Thirdly attempts were made to develop a negative selectable marker for homologous transformation based on the creation of mutants defective in uracil or acetate utilisation and their restoration to wild-type phenotype by transformation with the wild-type gene. Finally the use of antisense expression was investigated. An antisense section of the Chlamydomonas nuclear gene encoding oxygen evolving complex 1 was inserted into the genome of Chlamydomonas under the control of a strong Chlamydomonas promoter and the resulting transformants analysed for phenotypic effects and the presence of an antisense transcript

Transcriptome analysis of root transporters reveals participation of multiple gene families in the response to cation stress.

Plant nutrition critically depends on the activity of membrane transporters that translocate minerals from the soil into the plant and are responsible for their intra- and intercellular distribution. Most plant membrane transporters are encoded by multigene families whose members often exhibit overlapping expression patterns and a high degree of sequence homology. Furthermore, many inorganic nutrients are transported by more than one transporter family. These considerations, coupled with a large number of so-far non-annotated putative transporter genes, hamper our progress in understanding how the activity of specific transporters is integrated into a response to fluctuating conditions. We designed an oligonucleotide microarray representing 1096 Arabidopsis transporter genes and analysed the root transporter transcriptome over a 96-h period with respect to 80 mm NaCl, K+ starvation and Ca2+ starvation. Our data show that cation stress led to changes in transcript level of many genes across most transporter gene families. Analysis of transcriptionally modulated genes across all functional groups of transporters revealed families such as V-type ATPases and aquaporins that responded to all treatments, and families – which included putative non-selective cation channels for the NaCl treatment and metal transporters for Ca2+ starvation conditions – that responded to specific ionic environments. Several gene families including primary pumps, antiporters and aquaporins were analysed in detail with respect to the mRNA levels of different isoforms during ion stress. Cluster analysis allowed identification of distinct expression profiles, and several novel putative regulatory motifs were discovered within sets of co-expressed genes