Changes in the levels of polyamines are correlated with the activation or repression of developmental response pathways, but the role of polyamine transporters in the regulation of polyamine homeostasis and thus indirectly gene expression, has not been previously addressed. Here we show that the A. thaliana and rice transporters AtPUT5 and OsPUT1 were localized to the ER, while the AtPUT2, AtPUT3, and OsPUT3 were localized to the chloroplast by transient expression in N. benthamiana. A. thaliana plants that were transformed with OsPUT1 under the control the PUT5 promoter were delayed in flowering by 16days. In contrast, put5 mutants flowered four days earlier than WT plants. The delay of flowering was associated with significantly higher levels of spermidine and spermidine conjugates in the leaves prior to flowering. A similar delay in flowering was also noted in transgenic lines with constitutive expression of either OsPUT1 or OsPUT3. All three transgenic lines had larger rosette leaves, thicker flowering stems, and produced more siliques than wild type plants. In contrast, put5 plants had smaller leaves, thinner flowering stems, and produced fewer siliques. Constitutive expression of PUTs was also associated with an extreme delay in both plant senescence and maturation rate of siliques. These experiments provide the first genetic evidence of polyamine transport in the timing of flowering, and indicate the importance of polyamine transporters in the regulation of flowering and senescence pathways.
Agrobacterium rhizogenes transformation is a more rapid method of obtaining transgenic and edited rubber dandelion (Taraxacum kok-saghyz) plants than Agrobacterium tumefaciens. The hairy root rol genes are present alongside transgenes after transformation, and they change the morphology of rubber dandelion significantly. Although these rol genes are useful visual markers indicating successful transformation of rubber dandelion, they modify the phenotype induced by the target transgenes and are ultimately detrimental to agronomic traits. Fortunately, the rol genes can be removed by conventional plant breeding because they segregate in progeny separately from the targeted transgenes. However, it is preferable to have preliminary identification of promising effects induced by transgenes or gene edits before rol gene removal so that only the best plants are used for breeding. Therefore, the goal of this research was to characterize rol– and rol+ plant morphology so that, in the future, rol+ transgene+ plants can be easily distinguished from rol+ transgene– plants. This requires that rol gene–induced morphological changes and simply assayed physiological traits are first characterized thoroughly so that transgene changes may be observed. Taproot formation is reduced or eliminated in rubber dandelion by rol genes, and rol-induced hairy roots are identifiable easily because they grow shallowly in potting soil, so only partial unearthing is needed. Both leaf and flower numbers are increased by rol genes, but leaves and flowers are smaller than in rubber dandelion wild type with longer stalks. The rosette doming phenotype caused by the induction of a large number of leaf primordia is obvious in rooted plants as young as 1 month old. Photosynthetic rates are reduced significantly in rol+ plants, although growth is not. An accurate description of the morphology of rubber dandelion after A. rhizogenes transformation may allow for initial selection of promising transformed plants before confirmation with polymerase chain reaction, by phenotypic comparison of plants expressing transgenes and the rol gene, with those only expressing the rol gene.
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