Localisation of Abundant and Organ-Specific Genes Expressed in Rosa hybrida Leaves and Flower Buds by Direct In Situ RT-PCR

In situ PCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues. In situ PCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants, in situ RT-PCR is widely used in the expression localisation of specific genes, including MADS-box and other function-specific genes or housekeeping genes in floral buds and other organs. This method is especially useful in small organs or during early developmental stages when the separation of particular parts is impossible. In this paper, we compared three different labelling and immunodetection methods by using in situ RT-PCR in Rosa hybrida flower buds and leaves. As target genes, we used the abundant β-actin and RhFUL gene, which is expressed only in the leaves and petals/sepals of flower buds. We used digoxygenin-11-dUTP, biotin-11-dUTP, and fluorescein-12-dUTP-labelled nucleotides and antidig-AP/ streptavidin-fluorescein-labelled antibodies. All of the used methods gave strong, specific signal and all of them may be used in localization of gene expression on tissue level in rose organs

Manipulation of MKS1 gene expression affects Kalanchoe blossfeldiana and Petunia hybrida phenotypes

The establishment of alternative methods to chemical treatments for growth retardation and pathogen protection in ornamental plant production has become a major goal in recent breeding programmes. This study evaluates the effect of manipulating MAP kinase 4 nuclear substrate 1 (MKS1) expression in Kalanchoe blossfeldiana and Petunia hybrida. The Arabidopsis thaliana MKS1 gene was overexpressed in both species via Agrobacterium-mediated transformation, resulting in dwarfed phenotypes and delayed flowering in both species and increased tolerance to Pseudomonas syringae pv. tomato in transgenic Petunia plants. The lengths of the stems and internodes were decreased, while the number of nodes in the transgenic plants was similar to that of the control plants in both species. The transgenic Kalanchoe flowers had an increased anthocyanin concentration, and the length of the inflorescence stem was decreased. The morphology of transgenic Petunia flowers was not altered. The results of the Pseudomonas syringae tolerance test showed that Petunia plants with one copy of the transgene reacted similarly to the nontransgenic control plants; however, plants with four copies of the transgene exhibited considerably higher tolerance to bacterial attack. Transgene integration and expression was determined by Southern blot hybridization and RT-PCR analyses. MKS1 in wild-type Petunia plants was down-regulated through a virus-induced gene silencing (VIGS) method using tobacco rattle virus vectors. There were no significant phenotypic differences between the plants with silenced MKS1 genes and the controls. The relative concentration of the MKS1 transcript in VIGS-treated plants was estimated by quantitative RT-PCR

Flowering conditions affect flower longevity in Syringa vulgaris and cause changes in protein content, protease activity and expression of a KDEL-CysEP gene

Forcing is a method that is usually used to induce flowering in plants, independent of the natural blooming period. The temperatures required to start blooming in lilac in November are ca. 37°C causing degeneration of flowers. Forcing at 15 °C in November requires 49 days to bloom as compared to 23 days for the standard 37 °C, but gives panicles of much better quality than those forced by standard method (37 °C). In this study, we have investigated the protein content, total protease activity, and cysteine protease activity at different stages of flowering (flower bud whitening and swelling, open flowers, wilted flowers) for lilac flowers (Syringa vulgaris L., fam. Oleaceae) blooming under three different conditions: natural conditions in May and forcing in a greenhouse in November at 37 °C (standard forcing) or at 15 °C (alternative forcing). The protein content was relatively constant during flowering for each of the three sets of conditions. Flowers from 15 °C had a significantly lower total protease and cysteine endoprotease activity than flowers from 37 °C at all stages. Flowers from plants blooming in May had a very time-specific cysteine protease activity, which was dramatically higher for the open flower stage than for the other stages. The partial coding sequence for a KDEL-CysEP was isolated, and its expression was determined by qRT-PCR. The gene expression did not correlate with the cysteine endoprotease activity especially in May natural flowering and November alternative forcing at 15 °C. Alternative forcing method at 15 °C affected the flowering process delaying senescence, presumably due to the low cysteine protease activity. © 2016, The Author(s).National Center of Knowledge (NCN

Mechanical Stimulation Decreases Auxin and Gibberellic Acid Synthesis but Does Not Affect Auxin Transport in Axillary Buds; It Also Stimulates Peroxidase Activity in Petunia × atkinsiana

Thigmomorphogenesis (or mechanical stimulation-MS) is a term created by Jaffe and means plant response to natural stimuli such as the blow of the wind, strong rain, or touch, resulting in a decrease in length and an increase of branching as well as an increase in the activity of axillary buds. MS is very well known in plant morphology, but physiological processes controlling plant growth are not well discovered yet. In the current study, we tried to find an answer to the question if MS truly may affect auxin synthesis or transport in the early stage of plant growth, and which physiological factors may be responsible for growth arrest in petunia. According to the results of current research, we noticed that MS affects plant growth but does not block auxin transport from the apical bud. MS arrests IAA and GA3 synthesis in MS-treated plants over the longer term. The main factor responsible for the thickening of cell walls and the same strengthening of vascular tissues and growth arrestment, in this case, is peroxidase (POX) activity, but special attention should be also paid to AGPs as signaling molecules which also are directly involved in growth regulation as well as in cell wall modifications