FvWRKY48 binds to the pectate lyase FvPLA promoter to control fruit softening in Fragaria vesca.

The regulatory mechanisms that link WRKY gene expression to fruit ripening are largely unknown. Using transgenic approaches, we showed that a WRKY gene from wild strawberry (Fragaria vesca), FvWRKY48, may be involved in fruit softening and ripening. We showed that FvWRKY48 is localized to the nucleus and that degradation of the pectin cell wall polymer homogalacturonan, which is present in the middle lamella and tricellular junction zones of the fruit, was greater in FvWRKY48-OE (overexpressing) fruits than in empty vector (EV)-transformed fruits and less substantial in FvWRKY48-RNAi (RNA interference) fruits. Transcriptomic analysis indicated that the expression of pectate lyase A (FvPLA) was significantly downregulated in the FvWRKY48-RNAi receptacle. We determined that FvWRKY48 bound to the FvPLA promoter via a W-box element through yeast one-hybrid, electrophoretic mobility shift, and chromatin immunoprecipitation quantitative polymerase chain reaction experiments, and ß-glucosidase activity assays suggested that this binding promotes pectate lyase activity. In addition, softening and pectin degradation were more intense in FvPLA-OE fruit than in EV fruit, and the middle lamella and tricellular junction zones were denser in FvPLA-RNAi fruit than in EV fruit. We speculated that FvWRKY48 maybe increase the expression of FvPLA, resulting in pectin degradation and fruit softening.

Nicotine concentration in leaves of flue-cured tobacco plants as affected by removal of the shoot apex and lateral buds.

It is believed that the nicotine concentration in tobacco is closely correlated with the amount of nitrogen (N) supplied. On the other hand, N uptake mainly occurs at the early growth stage, whereas nicotine concentration increases at the late growth stage, especially after removing the shoot apex. To identify the causes of the increased nicotine concentration in tobacco plants, and to compare the effects of different ways of mechanical wounding on nicotine concentration, field experiments were carried out in Fuzhou, Fujian Province in 2003 and 2004. Excision of the shoot apex had almost no influence on N content in the plant; however, it caused dramatic increases in nicotine concentration in leaves, especially in the middle and upper leaves. An additional increase of the nicotine concentration was obtained by removal of axillary buds. The wounding caused by routine leaf harvests, however, did not change the leaf nicotine concentration, and neither did reducing leaf harvest times. The present results revealed no direct relationship between N supply and nicotine concentration in tobacco leaves, and indicate that not all kinds of mechanical wounding were capable of stimulating nicotine synthesis in tobacco plants. Since nicotine production is highly dependent on the removal of apical meristems and hence on the major sources of auxin in the plant, and application of 1-naphthylacetic acid onto the cut surface of the stem after removing the shoot apex markedly decreased the nicotine concentration in different leaves and the total nicotine content in the plant, the results suggest that decreased auxin supply caused by removal of the shoot apex as a kind of mechanical wounding might regulate nicotine synthesis in the roots of tobacco plants.