Expression of Arabidopsis WEE1 in Tobacco Induces Unexpected Morphological and Developmental Changes

WEE1 regulates the cell cycle by inactivating cyclin dependent protein kinases (CDKs) via phosphorylation. In yeast and animal cells, CDC25 phosphatase dephosphorylates the CDK releasing cells into mitosis, but in plants, its role is less clear. Expression of fission yeast CDC25 (Spcdc25) in tobacco results in small cell size, premature flowering and increased shoot morphogenetic capacity in culture. When Arath;WEE1 is over-expressed in Arabidopsis, root apical meristem cell size increases, and morphogenetic capacity of cultured hypocotyls is reduced. However expression of Arath;WEE1 in tobacco plants resulted in precocious flowering and increased shoot morphogenesis of stem explants, and in BY2 cultures cell size was reduced. This phenotype is similar to expression of Spcdc25 and is consistent with a dominant negative effect on WEE1 action. Consistent with this putative mechanism, WEE1 protein levels fell and CDKB levels rose prematurely, coinciding with early mitosis. The phenotype is not due to sense-mediated silencing of WEE1, as overall levels of WEE1 transcript were not reduced in BY2 lines expressing Arath;WEE1. However the pattern of native WEE1 transcript accumulation through the cell cycle was altered by Arath;WEE1 expression, suggesting feedback inhibition of native WEE1 transcription

Sucrose metabolism during somatic and zygotic embryogeneses in Norway spruce : content of soluble saccharides and localisation of key enzyme activities

Changes in soluble carbohydrate compounds and their amounts were analysed during the somatic embryo maturation and seed development of Norway spruce (Picea abies (L.) Karst.). The data were correlated to the localisation of key enzymes of carbohydrate metabolism (invertase, EC 3.2.1.26; sucrose synthase, EC 2.4.1.13; phosphoglucomutase, EC 5.4.2.2). The decrease in total carbohydrate content as well as the accumulation of sucrose in later stages was a common feature in both systems. At the beginning of somatic embryo maturation the activity of sucrose synthase was low, and it gradually increased during development. At the final cotyledonary stage the SuSy localisation in somatic embryos resembled the pattern observed in cotyledonary stage zygotic embryos. Activity of invertase (pH 6.0) was detected in the early stages of somatic embryo development. Afterwards the activity decreased to almost undetectable levels in mature cotyledonary embryos. Very low invertase activity was detected during the period of seed development studied. The activity of phosphoglucomutase was high throughout the whole process of maturation in both zygotic and somatic embryos. The results are discussed in respect to the transition of developing embryos from metabolic to storage sink and the possible signalling potential of the changes

Comparing carbohydrate status during norway spruce seed development and somatic embryo formation

The carbohydrate status of developing seeds of Picea abies was examined in order to provide a frame of reference for the evaluation of changes in carbohydrate content in maturing somatic embryos of the same species. Samples were taken at weekly intervals from 12 May 1998 (estimated time of pollination) until 20 October 1998. The total non-structural carbohydrate content was high (≈150-180μg mg-1 dry weight) at the time of the first samples and the carbohydrate spectrum consisted of sucrose, glucose, fructose, and pinitol. A dramatic decrease in carbohydrate content took place from June 6 onwards, that was accompanied by changes in carbohydrate partitioning to favor sucrose over hexoses and the disappearance of pinitol. Raffinose and stachyose were first detected on July 28, and their content gradually increased thereafter. Isolated embryos and remaining megagametophytes were analyzed starting with September 1. Carbohydrate content was higher in isolated zygotic embryo than in the rest of the seed, with a slowly increasing fraction of raffinose and stachyose. Comparisons of presented data with the results of our previous study of somatic embryo carbohydrate status (Lipavská et al., 2000) revealed the following common features: (1) a decrease in total carbohydrate content and (2) an increase in sucrose:hexose ratios in developing seeds and embryonal suspensor mass. Marked differences were observed in carbohydrate spectra: (1) somatic embryo development was not accompanied by pinitol accumulation in any phase; (2) mature zygotic embryos, in contrast to mature somatic embryos, contained raffinose and stachyose. These observations will provide a solid basis for improvement of protocols for somatic embryogenesis in Picea