Arabidopsis emb175 and other ppr knockout mutants reveal essential roles for pentatricopeptide repeat (PPR) proteins in plant embryogenesis

Abstract

Pentatricopeptide repeat proteins (PPRPs) constitute one of the largest superfamilies in plants, with more than 440 identified in the Arabidopsis thaliana (L.) Heynh genome. While some PPRPs are known to take part in organelle gene expression, little is known about the broader biological contexts of PPRP gene function. Here, using developmental-and reverse-genetic approaches, we demonstrate that a number of PPRPs are essential early in plant development. We have characterized the Arabidopsis embryo-defective175 mutant and identified the EMB175 gene. Emb175 consistently displays aberrant cell organization and undergoes morphological arrest before the globular-heart transition. The emb175 mutation disrupts an intronless open reading frame encoding a predicted chloroplast-localized PPR protein-the first to be rigorously associated with an early embryo-lethal phenotype. To determine if other PPRP genes act in embryogenesis, we searched Arabidopsis insertion mutant collections for pprp knockout alleles, and identified 29 mutants representing 11 loci potentially associated with embryo-defective phenotypes. We assessed gene structures, T-DNA insertion position, and allelism for these loci and were able to firmly establish essential functions for six PPRP genes in addition to EMB175. Interestingly, Nomarski DIC microscopy revealed diverse embryonic defects in these lines, ranging from early lethality to dramatic late-stage morphological defects such as enlarged shoot apices and stunted cotyledons. Together, emb175 and these pprp knockout mutants establish essential roles for PPRPs in embryogenesis, thus broadening the known organismal context for PPRP gene function. The diversity of embpprp knockout phenotypes indicates that mutation of different PPRPs can, directly or indirectly, have distinct impacts on embryo morphogenesis.

Arabidopsis GLUTAMINE-RICH PROTEIN23Is Essential for Early Embryogenesis and Encodes a Novel Nuclear PPR Motif Protein That Interacts with RNA Polymerase II Subunit III

“…Several reports have shown that loss of function of the PPR gene in Arabidopsis often caused an embryo-lethal phenotype, although at relatively late stages of embryogenesis compared with grp23 (Tzafrir et al, 2004;Cushing et al, 2005). In addition, they also showed dramatic morphological defects, such as enlarged shoot apices and stunted cotyledons (Cushing et al, 2005). As we did not observe such defects in grp23 mutants as a result of its early embryo lethality, the role of GRP23 in meristematic cells remains to be addressed.…”

“…It is not clear whether this is an indirect or direct cause of the loss of function of GRP23. Several reports have shown that loss of function of the PPR gene in Arabidopsis often caused an embryo-lethal phenotype, although at relatively late stages of embryogenesis compared with grp23 (Tzafrir et al, 2004;Cushing et al, 2005). In addition, they also showed dramatic morphological defects, such as enlarged shoot apices and stunted cotyledons (Cushing et al, 2005).…”

“…Although several PPR genes have been shown to control cytoplasmic male sterility (Kazama and Toriyama, 2003) and fertility restoration (Bentolila et al, 2002;Brown et al, 2003;Desloire et al, 2003;Koizuka et al, 2003;Laforest et al, 2003;Akagi et al, 2004;Komori et al, 2004;Klein et al, 2005), it appears that the GRP23 function is not essential for gametophyte and endosperm development, which may partly be attributable to the genetic redundancy of PPR genes Cushing et al, 2005). Alternatively, there is a dosage effect for the gametophyte development for which only two or three cell divisions are required; they may receive sufficient GRP23 protein from their heterozygous maternal or paternal precursor cells.…”

“…of function of At3g49240 also displayed an embryo-defective phenotype (Cushing et al, 2005). At3g49240 encodes a predicted polypeptide of 629 amino acid residues containing 11 PPR motifs arranged in tandem.…”

“…Examples of mutants arrested very early during embryogenesis include fac1, a zygotic embryo-lethal mutant (Xu et al, 2005), rpn1a, a mutation in the RNP1 subunit of the 26S proteasome (Brukhin et al, 2005), titan mutants, in which genes proposed to be involved in chromosome maintenance and microtubule assembly are disrupted (Liu and Meinke, 1998;McElver et al, 2000;Tzafrir et al, 2002), and opt3, a mutation of a peptide transporter gene (Stacey et al, 2002). Interestingly, among the predicted EMB genes there are 17 putative pentatricopeptide repeat (PPR)-containing genes, of which 7 were shown to be essential for late embryogenesis (Tzafrir et al, 2004;Cushing et al, 2005).…”