Expression of Arabidopsis WEE1 in tobacco induces unexpected morphological and developmental changes

Ilario Siciliano,Dennis Francis,Golnaz Rafiei,Robert J Herbert,Natasha D Spadafora,Hana Ševčíková,M Beatrice Bitonti,Hilary J Rogers,Anne Lentz Grønlund,Helena Lipavská

doi:10.1038/s41598-019-45015-3

Ilario Siciliano, Dennis Francis + Show 8 more

Open Access

https://doi.org/10.1038/s41598-019-45015-3

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Abstract

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.

Highlights

The eukaryotic cell cycle is a conserved phosphorylation cascade in which key substrates require phosphorylation or dephosphorylation prior to the step in the cell cycle
Where the plant cell cycle diverges quite dramatically from other eukaryotes, is that Arabidopsis mutants deficient in WEE1 kinase grow and develop normally they are hypersensitive to DNA replication inhibitors such as hydroxyurea[10,22]
WT plants flowered when they had produced more than 20 leaves longer than 10 cm, while transgenic plants expressing Arath;WEE1 formed only around seven leaves of this size before they started to flower (Fig. 1c)

Summary

Introduction

The eukaryotic cell cycle is a conserved phosphorylation cascade in which key substrates require phosphorylation or dephosphorylation prior to the step in the cell cycle. Expression of the fission yeast CDC25 gene in both tobacco[15] and Arabidopsis[16], resulted in phenotypes that are consistent with its action in dephosphorylating and activating CDK. Consistent results were obtained in Arabidopsis plants expressing Spcdc2516, which showed a reduction in primary root length and increased production of lateral roots Another effect of Spcdc[25] expression in tobacco was precocious flowering with a dramatic reduction in both the time to flowering, and the number of leaves and nodes formed prior to flowering[20]. Where the plant cell cycle diverges quite dramatically from other eukaryotes, is that Arabidopsis mutants deficient in WEE1 kinase grow and develop normally they are hypersensitive to DNA replication inhibitors such as hydroxyurea[10,22]. WEE1 regulates CDK activity in a cell cycle dependent manner with a drop in WEE1 activity at the G2/M transition[23] and in both tobacco BY2 cells and in Arabidopsis roots, WEE1 protein is removed as cells enter mitosis via the 26 S proteasome

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