A CDC45 Homolog in Arabidopsis Is Essential for Meiosis, as Shown by RNA Interference–Induced Gene Silencing

Abstract

CDC45 is required for the initiation of DNA replication in yeast and cell proliferation in mammals and functions as a DNA polymerase ␣ loading factor in Xenopus . We have cloned a CDC45 homolog from Arabidopsis whose expression is upregulated at the G1/S transition and in young meiotic flower buds. One-third of Arabidopsis 35S : CDC45 T1 RNA interference lines are partially to completely sterile, and the proportion of sterile plants is increased by using a dmc1 promoter. T1 plants have decreased levels of the CDC45 transcript and contain 21-to 23-bp RNA fragments specific to the CDC45 gene. T2 transgenic lines, in which small RNA fragments are still present, were used to analyze S-phase entry by 5-bromodeoxyuridine incorporation, which was not altered compared with that in the wild type. However, microarray data show that other cell cycle genes are upregulated or downregulated. T2 plants also have highly reduced fertility. The severity of the phenotype is correlated with the levels of the CDC45 transcript and small RNA fragments. Severe chromosome fragmentation arising during meiosis, which is not the result of a defect in the repair of SPO11-induced double strand breaks, leads to abnormal chromosome segregation and defective pollen and ovule development.

TheArabidopsis-mei2-LikeGenes Play a Role in Meiosis and Vegetative Growth inArabidopsis

“…The chromosomal phenotypes first become apparent at postpachytene stages. Other Arabidopsis genes and mutants affecting meiosis for which defects are mainly seen after pachytene are dsy1 (Ross et al, 1997), dsy10 (Cai and Makaroff, 2001), mei1 (He et al, 1996;Grelon et al, 2003), mmd1/duet (Reddy et al, 2003;Yang et al, 2003), At CAPE1, At CAPE2 (Siddiqui et al, 2003), and At CDC45 (Stevens et al, 2004). Several of these encode proteins that are directly implicated in chromatin organization and include proteins that are considered to act in mitosis as well as meiosis.…”

Mre11 Deficiency in Arabidopsis Is Associated with Chromosomal Instability in Somatic Cells and Spo11-Dependent Genome Fragmentation during Meiosis

“…The chromosome fragmentation in such mutants is likely of premeiotic origin because it does not depend on Spo11 (Grelon et al, 2003;Stevens et al, 2004). In contrast with mei1 and cdc45 mutants, most of the genome fragmentation was suppressed by the spo11-1 mutation in mre11-3 plants.…”

Regulating DNA Replication in Plants

“…In spite of these similarities, its role in plant DNA replication has not been shown. Arabidopsis CDC45 is unable to complement a yeast cdc45 temperature-sensitive mutant and Arabidopsis RNAi plant lines with reduced CDC45 transcripts did not show defects in DNA synthesis (Stevens et al 2004).…”

“…Like in yeast and mammalian cells, Arabidopsis CDC45 gene expression is cell cycle regulated, showing high levels at the G 1 /S transition (Stevens et al 2004). In spite of these similarities, its role in plant DNA replication has not been shown.…”