DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis

Abstract

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1’s primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

ArabidopsisRAN GTPases are critical for mitosis during male and female gametogenesis

“…RANGAP1, RCC1, importins, and so on. RAN1 and DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) co-localize transiently during meiosis to mediate cell division and gamete viability [49]. In rice (Oryza sativa), OsRAN1overexpressed lines contained more proliferating cells, especially under cold stress, implying a role of RAN1 Because male and female gametophytes develop within multiple layers of somatic cells, imaging mitosis during male and female gametophytic development is challenging, if possible.…”

“…RANGAP1, RCC1, importins, and so on. RAN1 and DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) co‐localize transiently during meiosis to mediate cell division and gamete viability [49]. In rice ( Oryza sativa ), OsRAN1 ‐overexpressed lines contained more proliferating cells, especially under cold stress, implying a role of RAN1 in mitosis and cell cycle progression [50].…”

DEFECTIVE EMBRYO AND MERISTEMS1 (DEM1) Is Essential for Cell Proliferation and Cell Differentiation in Tomato

“…Genome sequence analysis has revealed that dicotyledonous plants such as tomato and Arabidopsis encode two distinct copies of the DEFECTIVE EMBRYO AND MERISTEMS ( DEM ) gene with the DEM1 and DEM2 loci encoding a highly similar protein of unknown biochemical function, except that the encoded proteins interact with RAS-LIKE NUCLEAR PROTEIN (RAN) [ 41 , 42 ]. RAN is conserved in all eukaryotes and plays a fundamental role in nucleus–cytoplasm transport and cell division.…”

“…Furthermore, the degree of defective SAM and RAM development in the tomato dem1 mutant resulted in this plant line failing to proceed past cotyledon emergence as part of the seedling stage of early vegetative development [ 41 ]. In contrast to the tomato dem1 mutant, the Arabidopsis dem1 and dem2 single mutants express wild-type-like phenotypes throughout their vegetative development [ 42 ]. However, Arabidopsis plants carrying a single functional copy of the DEM1 gene, and which are homozygous for mutation of the DEM2 gene (specifically, DEM1/dem1 dem2/dem2 plants), showed defects in meiotic cell division and gamete viability [ 42 ].…”

“…In contrast to the tomato dem1 mutant, the Arabidopsis dem1 and dem2 single mutants express wild-type-like phenotypes throughout their vegetative development [ 42 ]. However, Arabidopsis plants carrying a single functional copy of the DEM1 gene, and which are homozygous for mutation of the DEM2 gene (specifically, DEM1/dem1 dem2/dem2 plants), showed defects in meiotic cell division and gamete viability [ 42 ]. Repeated attempts to generate an Arabidopsis dem1 dem2 double mutant via a standard genetic crossing approach have failed, which is consistent with an essential role for DEM proteins in cell division and/or gamete viability [ 42 ].…”