Potato life-cycle : from genes to promoters to cis-acting elements

Potato plants undergo several developmental stages during their life-cycle, including stolon formation, tuber initiation, tuber growth, dormancy and sprouting. Most of these stages are continuous processes, meaning that different developmental stages can occur simultaneously in one plant. Therefore, in order to study the different stages of stolon and tuber development a synchronised system was considered necessary. Using an in vitro synchronised system plant material was collected at 20 time points resulting in 22 different samples. To monitor gene expression during all these stages the cDNA-AFLP technique was used on the 22 templates, as well as fifteen different plant tissues and 10 samples where tuberisation was inhibited with gibberellins (GA 4+7 ). In total nearly 2,000 transcript derived fragments (TDF's) were monitored, and 116 TDF's corresponding to genes differentially expressed during the potato life-cycle were isolated and sequenced. The sequence similarities between these TDF's and the sequences in the databases indicated that most of the genes are involved in defense, stress, storage and signal transduction pathways. Analysis of the tissue specificity of the 1,926 TDF's revealed that one third were exclusively expressed in stolons and/or tubers, flowers and/or fruits, and that only 1.5% were common among stem (like) tissues such as stems, stolons and/or tubers. Based on the differential expression during the potato life-cycle, tissue specificity, influence of GA and homology to relevant genes, 33 TDF's were selected to proceed for promoter isolation. Two TDF's, TDFL511 and TDF1041, were used as probes to isolate the corresponding promoter(s), one for TDFL511 and two for TDF1041, by screening a potato genomic DNA library. TDFL511 showed similarity to alcohol dehydrogenases and its corresponding protein, Stgan, is likely to be involved in the biosynthesis or breakdown of compounds affecting GA levels in the plant. The promoter region of this gene was fused to a reporter gene encoding the β-glucoronidase (GUS) protein and introduced into potato plants. Both GUS staining and in situ hybridisation experiments revealed that Stgan was exclusively expressed in the stolon cortex, in parenchyma cells. TDF1041 is highly homologous to genes encoding non-specific lipid transfer proteins (nsLTP), which are capable of binding lipid compounds in plant tissues. Promoter- gus gene fusions revealed that one promoter is specifically expressed in phloem cells mostly in young plants and tissues, and the other promoter showed little tissue or organ specificity. These differences in expression are likely to be due to a 331 bp insertion present in the tissue specific promoter. The representativeness in the tetraploid potato genome of the remaining 31 TDF's was determined with Southern blot analysis. Specific primers were designed for the 14 TDF's with low copy number (maximum 3 copies or alleles) and the corresponding putative promoter regions were isolated by genome walking. The putative promoter corresponding to TDFL431, with high similarity to copper chaperones for Cu/Zn superoxide dismutases (CCS), was fused with the firefly luciferase gene. StCCS, the gene corresponding to TDFL431, was found mostly in the cortex of stem (like) tissues, at a relatively high level in the stem nodes, stolons and tubers. In addition, StCCS revealed to be induced by auxin, fructose, sucrose and glucose, and inhibited by high concentrations of copper. In order to identify specific cis-acting elements in the promoter regions of the 14 (putative) potato promoters, a computer program was developed and named PRECISE ( P rediction of RE gulatory CIS -acting E lements). Using this software package about 1,400 putative cis-elements between 6 and 16 nucleotides long, common to at least 8 of the 14 promoters, were identified. Comparison of these oligomers to all the previously characterised cis-elements resulted in the identification of nine specific motifs, which are influenced by factors that play an important role during potato tuber life-cycle such as light, sugars, GA, auxin and abscisic acid. In order to test the potentialities of PRECISE to identify cis-elements, a set of 64 promoters driving 19 different genes (or gene families) involved in starch biosynthesis in Arabidopsisthaliana were analysed. To select the most relevant oligomers out of the more than 10,000 putative motifs found with PRECISE an Arabidopsis oligomers database (AROLI) was set up and included all the oligomers between 3 and 11 nucleotides and their frequencies in Arabidopsis non-coding regions (excluding introns). Comparing the twenty most significant motifs to the ones present in PLACE database resulted in seventeen motifs, which were highly homologous to a part or the entire sequences of previously characterised cis-elements. These results proved the power of the PRECISE software package in the prediction of promoter cis-acting elements.