Work package 1: Characterisation of protein-protein interactions of putative components of the complex that controls tuberization.
Using yeast two-hybrid (Y2H) assays we demonstrated that the StCEN protein binds to the complex that controls tuberization (the tuberigen activation complex (TAC)). These results were confirmed using bimolecular fluorescence complementation. We conclude that the negative tuberization (StCEN) competes with the positive tuberization component (SP6A) and that when StCEN forms a complex the tuberigen complex is no longer active.
Work package 2: The detailed expression pattern of TFL1
Our discovery that StCEN is a powerful negative regulator of both tuberisation and post-harvest tuber sprouting provides new insights into how the tuber life cycle is regulated. To add to this knowledge, we aimed to acquire detailed understanding of the temporal and spatial expression pattern of StCEN and its response to environmental stimuli. We used two approaches to investigate the expression pattern of StCEN- in situ hybridisation and a StCEN promoter reporter (Green Fluorescent
Protein) construct. Preliminary results, using both methods, indicate StCEN expression in stolons in a distinct cell layer, external to vascular strands. The expression is present just before stolon swelling and in the region of swelling tissue as the tuber initiates. StCEN1 expression appears to be initially in the cortical cambium; the tissue that divides for tuber swelling. As tubers develop, expression is throughout dividing, swelling tissue.
Work package 3: Transcriptional profiles associated with altered StCEN expression in stolons from transgenic lines
To identify potential TAC targets, we carried out transcriptomic analysis of non-swelling stolons from WT compared with RNAi lines silenced for StCEN. As the StCEN RNAi lines tuberise earlier, we reasoned that the non-swelling stolons from this genotype are primed for tuberisation and so a comparison with WT should reveal genes expressed early in tuber initiation.
Based on prior literature, we selected genes that had been associated with early tuberization and show that these genes are in fact up regulated in the RNAi lines compared with WT and OE lines. More remarkably, in the set of strongly up-regulated genes in cluster 2 we identified several transcripts encoding for germin like proteins, described in Arabidopsis as plasmodesmata-located. Transcripts annotated as germins are expressed at levels between ca. 200 and 700-fold higher in the stolons from RNAi lines than in those from controls suggesting that this family of proteins may have a relevant role in the switch in from apoplastic to symplastic sucrose unloading, marking initiation of tubers.
Work package 4: Response of TFL1 transgenic lines to abiotic stress
StCEN transgenic lines of S. tuberosum cv Desiree were tested for tolerance to heat stress in a heated glasshouse. Wild type (WT) Desiree was compared with RNAi and OE lines. For plants subjected to heat stress, the tuber yield of WT plants was significantly less than for RNAi lines 28 and 31, whereas for OE lines 2 and 7, there was a trend towards lower yield than in WT plants. These preliminary data support the hypothesis that decreased expression of StCEN can offer protection against tuber yield reduction at elevated temperature however this conclusion remains tentative until there is further confirmation with additional transgenic lines.
Work package 5: Allelic variation in TFL1 and StCDF1
Previously we observed wide variation in tuber initiation time in the diploid 06H1 population despite our observation that there is no segregation for foliage senescence at the well documented StCDF-1 locus on chromosome 5. Based on our preliminary evidence we believe polymorphisms in the StCEN locus account for this variation and we now wish to identify which alleles of StCEN are associated with variation for tuber initiation in this and related populations.
We developed PCR-based markers (Cleaved amplified polymorphic sequence (CAPS)) for the four StCEN alleles identified in the 06H1 parents and used these to determine the precise allelic composition of StCEN in ca. 180 genotypes from this population. An initial experiment, based on tuberization of whole plants indicated significant variation between the four genotype classes. However, the large variation between replicates indicated by large standard errors demonstrates that many more replicates of each genotype is required to obtain statistically significant results. Nevertheless, these preliminary results provide encouraging results that StCEN allelic variation underpins variation in tuberization time at least in the 06H1 population.
Work package 6:Results of WP1 andWP3 are included in the following open access publication.
Zhang, X. et al.,(2020). TERMINAL FLOWER‐1/CENTRORADIALIS inhibits tuberisation via protein interaction with the tuberigen activation complex. The Plant Journal, 103(6), 2263-2278.https://doi.org/10.1111/tpj.14898
