TheArabidopsis thalianaCheckpoint Kinase WEE1 Protects against Premature Vascular Differentiation during Replication Stress

Abstract

A sessile lifestyle forces plants to respond promptly to factors that affect their genomic integrity. Therefore, plants have developed checkpoint mechanisms to arrest cell cycle progression upon the occurrence of DNA stress, allowing the DNA to be repaired before onset of division. Previously, the WEE1 kinase had been demonstrated to be essential for delaying progression through the cell cycle in the presence of replication-inhibitory drugs, such as hydroxyurea. To understand the severe growth arrest of WEE1-deficient plants treated with hydroxyurea, a transcriptomics analysis was performed, indicating prolonged S-phase duration. A role for WEE1 during S phase was substantiated by its specific accumulation in replicating nuclei that suffered from DNA stress. Besides an extended replication phase, WEE1 knockout plants accumulated dead cells that were associated with premature vascular differentiation. Correspondingly, plants without functional WEE1 ectopically expressed the vascular differentiation marker VND7, and their vascular development was aberrant. We conclude that the growth arrest of WEE1-deficient plants is due to an extended cell cycle duration in combination with a premature onset of vascular cell differentiation. The latter implies that the plant WEE1 kinase acquired an indirect developmental function that is important for meristem maintenance upon replication stress.

The plant‐specific CDKB 1‐ CYCB 1 complex mediates homologous recombination repair in Arabidopsis

“…For this analysis, wee1 was used as a positive control and, consistent with previous data, was found to be highly sensitive to HU, whereas it shows no growth abnormalities on control medium (De Schutter et al , 2007; Cools et al , 2011). In contrast, root growth on HU of all tested cycb1 mutants was comparable to the growth of wild‐type plants (Fig 1C and D).…”

“…As a positive control, we used mutants in ku70 that were shown to grow as the wild type on medium without drugs (Cools et al , 2011). Whereas ku70 mutants were sensitive to BLM and grew only very little consistent with previous reports (Tamura et al , 2002; West et al , 2002; Cools et al , 2011), no significant difference was found between cycb1 single and double mutants versus the wild type again with the exception of cycb1;1 cycb1;2 (Fig 1E and F). Comparing root growth ratios on plates with and without BLM indicated that cycb1;1 cycb1;2 is also not hypersensitive to this drug (Fig EV3A).…”

“…While homologs of ATM and its sister kinase ATR (ATM‐ and Rad3‐related) predominantly involved in replication stress response by sensing single‐stranded DNA have also been identified in Arabidopsis (Garcia et al , 2000; Culligan et al , 2004; Culligan & Britt, 2008), no homologs of Chk2 or its sister kinase Chk1 could be found in plants to date. Furthermore, even though a homolog of the yeast Wee1 kinase exists in Arabidopsis and other plants, its function appears to be different as Arabidopsis WEE1 was found to act during S phase after hydroxyurea (HU)‐induced replication stress and not in repressing CDK activity during mitosis or blocking cell division after DSB formation (De Schutter et al , 2007; Cools et al , 2011). Moreover, transgenic plants expressing a mutant version of CDKA;1, the Arabidopsis homolog of mammalian Cdk1 and Cdk2, in which the putative WEE1 target sites were replaced with non‐phosphorylatable amino acids, were not hypersensitive to HU indicated that cell‐cycle arrest after DNA damage is differently regulated in plants (Dissmeyer et al , 2009, 2010).…”

SUPPRESSOR OF GAMMA RESPONSE1 Links DNA Damage Response to Organ Regeneration

“…CDKB1;2, CDKB2;1, and KNOLLE [Yoshiyama et al, 2009;Missirian et al, 2014]), while directly up-regulating other factors (e.g. CYCB1;1, SMR-5,7 [Weimer et al, 2016a;Yi et al, 2014], and WEE1 indirectly [De Schutter et al, 2007, Cools et al, 2011). IR-induced SOG1 also transcriptionally activates DNA repair genes, including BRCA1 and RAD51 (Yoshiyama et al, 2009), which function in homologous recombination (HR)-based repair in S and G2 phase during normal growth (Shrivastav et al, 2008;Menges et al, 2003).…”

“…HR repair can help to restore a damaged cell's genomic integrity in addition to the faster (Mao et al, 2008), predominating canonical nonhomologous end-joining repair pathway (Čermák et al, 2017). SOG1's induction of HR repair, in combination with cell cycle arrest, can prevent cell death from mitosis occurring prematurely in the presence of unrepaired DSBs (Furukawa et al, 2010;Cools et al, 2011;Léguillier et al, 2012;Yi et al, 2014).…”