Investigating the Role of Cytokinin and Beta-1,3-Glucanases in Potato Tuber Dormancy and Sprouting
Year: 2012
Type: dissertation
Abstract: Potato tubers are mainly consumed fresh, generating an all year demand only to be met by long-term storage of tubers. Sprouting of tubers is an important factor for quality loss during storage, with tubers shrivelling from remobilization of storage substances and water loss. Unravelling the molecular mechanisms underlying potato tuber dormancy and sprouting is therefore of interest for both breeders and processing industries and may enable directed manipulation of dormancy length. Phytohormone measurements and treatment experiments have credited cytokinins (CKs) with a role in dormancy break. To further elucidate the role of this phytohormone, transgenic plants with altered CK biosynthesis were generated by expressing either an isopentenyltransferase (IPT) from Agrobacterium tumefaciens or an Arabidopsis thaliana CK oxidase/ dehydrogenase (CKX1) gene and analysed in detail. Strong expression of IPT, the rate-limiting enzyme of CK biosynthesis, led to bushy growth, small leaflets, inhibition of root growth and limited or absent tuber formation. Oone weakly expressing line (IPT-6), which resembled wild-type (WT) concerning plant growth and tuber yield, formed thicker and longer tuber sprouts compared to WT. In an in vitro sprouting assay (SRA), IPT-6 tubers were also shown to sprout earlier than the WT. In contrast, when CK content was lowered by expression of CKX1, transgenic plants showed reduced shoot growth, an increased number of fragile side shoots and a lower number of leaflets per leaf. CKX1 transcript abundance was inversely correlated to tuber yield and onset of sprouting. This effect was most pronounced in the strongest line (CKX1-4) which sprouted up to eight weeks later than the WT. Moreover, sprouts of CKX1-4 tubers remained diminutive after bud break and failed to initiate proper growth. In an in vitro sprouting assay using WT and CKX1-4 tubers, GA3 treatment did not initiate sprouting in the transgenic tubers. Additional transcriptional analysis of WT, IPT-6 and CKX1-4 tubers revealed that many transcripts up-regulated during GA3-mediated sprouting in WT and IPT-6 are not induced in CKX1-4 tubers. Together, these results clearly demonstrated that the presence of cytokinins is an essential requirement for the induction of tuber sprouting. Experiments with fluorescent dyes and markers have shown that meristematic cells in potato tubers are symplasmically isolated from the surrounding tissue during dormancy. In other species, symplasmic isolation was shown to result from callose deposition at plasmodesmata (PD), symplasmic connections between adjacent plant cells. To investigate whether callose deposition is an important factor of dormancy control, 34 members of the beta-1,3-glucanase (B13G) gene family encoding callose-degrading enzymes were identified in the potato genome. Comparison of available microarray data revealed several candidate genes which were up-regulated shortly before or at sprouting. Two of these candidates, termed StPdB13G_1 and StPdB13G_2, were shown to have high protein sequence similarity to a PD-located beta-1,3-glucanase of Arabidopsis thaliana (AtBG_PAP). Transgenic plants carrying an RNAi construct to silence both genes showed no phenotypical differences to the WT and during post-harvest storage, their tubers sprouted slightly, but not significantly later than WT tubers. To investigate the effects of the over-expression of a PD-located glucanase, AtBG_PAP was expressed in transgenic potatoes. This resulted in a WT-like plant phenotype and a delay in sprouting of about one week. Taken together, de-regulation of PD-glucanases seemed not to contribute greatly to tuber dormancy release. Additional evidence for this was provided by immunolocalisation of callose in WT tubers which showed no differences in callose distribution before, during and after onset of sprouting. Instead, massive formation of new vascular tissue was observed and analysis of transcription data revealed up-regulation of auxin biosynthesis and signalling pathway genes as well as markers for vascular development. These findings indicate that de novo formation of vascular bundles may support carbon allocation to meet the energy demand of cells in the newly forming sprout.
Author: Anja Hartmann
Language: English
Subfield: Plant Science
Domain: Life Sciences
SDG: Zero hunger
Open Access status: green
OpenAlex ID: W3158406738