Plastid encoded RNA polymerase activity and expression of photosynthesis genes required for embryo and seed development in Arabidopsis.

Dmitry Kremnev,Ã…Sa Strand

doi:10.3389/fpls.2014.00385

Abstract

Chloroplast biogenesis and function is essential for proper plant embryo and seed development but the molecular mechanisms underlying the role of plastids during embryogenesis are poorly understood. Expression of plastid encoded genes is dependent on two different transcription machineries; a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. However, the division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear. We show here that PLASTID REDOX INSENSITIVE 2 (PRIN2) and CHLOROPLAST STEM-LOOP BINDING PROTEIN 41 kDa (CSP41b), two proteins identified in plastid nucleoid preparations, are essential for proper plant embryo development. Using Co-IP assays and native PAGE we have shown a direct physical interaction between PRIN2 and CSP41b. Moreover, PRIN2 and CSP41b form a distinct protein complex in vitro that binds DNA. The prin2.2 and csp41b-2 single mutants displayed pale phenotypes, abnormal chloroplasts with reduced transcript levels of photosynthesis genes and defects in embryo development. The respective csp41b-2prin2.2 homo/heterozygote double mutants produced abnormal white colored ovules and shrunken seeds. Thus, the csp41b-2prin2.2 double mutant is embryo lethal. In silico analysis of available array data showed that a large number of genes traditionally classified as PEP dependent genes are transcribed during early embryo development from the pre-globular stage to the mature-green-stage. Taken together, our results suggest that PEP activity and consequently the switch from NEP to PEP activity, is essential during embryo development and that the PRIN2-CSP41b DNA binding protein complex possibly is important for full PEP activity during this process.

Highlights

The chloroplasts house the photosynthetic light reactions where sunlight is converted into chemical energy
Our results suggest that plastid-encoded bacterial-type RNA polymerase (PEP) activity and the switch from nuclear-encoded phage-type RNA polymerase (NEP) to PEP activity, is essential during embryo development and that the PLASTID REDOX INSENSITIVE 2 (PRIN2)-CSP41b protein complex potentially is important for full PEP activity during this process
Full length PRIN2 protein fused to a cMyc-tag was expressed in Arabidopsis plants, intact chloroplasts were isolated and PRIN2-containing protein complexes were precipitated with anti-cMyc antibody, proteins were separated on SDS PAGE and distinct bands identified using mass spectrometry (Figure 1A)

Summary

Introduction

The chloroplasts house the photosynthetic light reactions where sunlight is converted into chemical energy. Abortion of developing embryos is known to occur when amino acid, nucleotide or fatty acid biosynthesis is impaired, or when import of chloroplast proteins and translation are disrupted (McElver et al, 2001; Tzafrir et al, 2003; Hsu et al, 2010; Bryant et al, 2011). Chloroplasts are detected as early as at the globular stage of the embryo (Tejos et al, 2010) and transcript profiling during embryo development showed a significant increase in the expression of nuclear genes encoding components involved in energy production, carbon fixation and photosynthesis already from the globular embryogenic stage (Spencer et al, 2007; Le et al., 2010; Belmonte et al, 2013). It was shown that Brassica embryos were able to fix CO2 and contributing to embryo growth rate and biomass (Goffman et al, 2005)

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