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

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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 emb–pprp knockout phenotypes indicates that mutation of different PPRPs can, directly or indirectly, have distinct impacts on embryo morphogenesis.

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Abbreviations

EMB :

Embryo-defective

DIC:

Differential interference contrast

I-PCR:

Inverse polymerase chain reaction

ORF:

Open reading frame

PPR:

Pentatricopeptide repeat

PPRP:

Pentatricopeptide repeat protein

RT-PCR:

Reverse-transcription-PCR

UTR:

Untranslated region

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Acknowledgements

We are grateful for support from the USDA (02-35304-12304 to DMV) and the Murdock Charitable Trust. Emb175 characterization was initiated with support from NSF-9604344 to DMV. The W.M. Keck Foundation provided support for digital imaging equipment. We thank Dr. David Meinke (Oklahoma State University) for emb175 complementation crosses with emb1899-1 and −2 when those mutants were identified in the course of this work. cDNAs and mutant seed stocks were provided by the ABRC. We thank Tovi M. Anderson and John E. Houghland for assistance.

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  1. Department of Biology, and Program in Biochemistry, Biophysics and Molecular Biology, Whitman College, Walla Walla, WA, 99362, USA

    Daniel A. Cushing, Nancy R. Forsthoefel, Daniel R. Gestaut & Daniel M. Vernon

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  1. Daniel A. Cushing
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  2. Nancy R. Forsthoefel
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  3. Daniel R. Gestaut
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  4. Daniel M. Vernon
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Correspondence to Daniel M. Vernon.

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Cushing, D.A., Forsthoefel, N.R., Gestaut, D.R. et al. Arabidopsis emb175 and other ppr knockout mutants reveal essential roles for pentatricopeptide repeat (PPR) proteins in plant embryogenesis. Planta 221, 424–436 (2005). https://doi.org/10.1007/s00425-004-1452-x

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