Abstract
BackgroundTranscription of plastid-encoded genes requires two different DNA-dependent RNA polymerases, a nuclear-encoded polymerase (NEP) and plastid-encoded polymerase (PEP). Recent studies identified two related pfkB-type carbohydrate kinases, named FRUCTOKINASE-LIKE PROTEIN (FLN1 and FLN2), as components of the thylakoid bound PEP complex in both Arabidopsis thaliana and Sinapis alba (mustard). Additional work demonstrated that RNAi-mediated reduction in FLN expression specifically diminished transcription of PEP-dependent genes.ResultsHere, we report the characterization of Arabidopsis FLN knockout alleles to examine the contribution of each gene in plant growth, chloroplast development, and in mediating PEP-dependent transcription. We show that fln plants have severe phenotypes with fln1 resulting in an albino phenotype that is seedling lethal without a source of exogenous carbon. In contrast, fln2 plants display chlorosis prior to leaf expansion, but exhibit slow greening, remain autotrophic, can grow to maturity, and set viable seed. fln1 fln2 double mutant analysis reveals haplo-insufficiency, and fln1 fln2 plants have a similar, but more severe phenotype than either single mutant. Normal plastid development in both light and dark requires the FLNs, but surprisingly skotomorphogenesis is unaffected in fln seedlings. Seedlings genetically fln1-1 with dexamethasone-inducible FLN1-HA expression at germination are phenotypically indistinguishable from wild-type. Induction of FLN-HA after 24 hours of germination cannot rescue the mutant phenotype, indicating that the effects of loss of FLN are not always reversible. Examination of chloroplast gene expression in fln1-1 and fln2-1 by qRT-PCR reveals that transcripts of PEP-dependent genes were specifically reduced compared to NEP-dependent genes in both single mutants.ConclusionsOur results demonstrate that each FLN protein contributes to wild type growth, and acting additively are absolutely essential for plant growth and development.
Highlights
Transcription of plastid-encoded genes requires two different DNA-dependent RNA polymerases, a nuclear-encoded polymerase (NEP) and plastid-encoded polymerase (PEP)
FLN1 and FRUCTOKINASE-LIKE PROTEIN (FLN2) loss-of-function mutants have severe chlorotic phenotypes We took a reverse genetics approach and analyzed the available T-DNA insertion alleles to identify the role of each FLN in plant growth and development
FLN proteins are required for normal plastid development To investigate plastid development in fln1 and fln2 seedlings, we examined chloroplast ultrastructure in cotyledons of 7 day-old light-grown seedlings grown on Growth media (GM) by transmission electron microscopy (TEM) (Figure 6)
Summary
Transcription of plastid-encoded genes requires two different DNA-dependent RNA polymerases, a nuclear-encoded polymerase (NEP) and plastid-encoded polymerase (PEP). Additional work demonstrated that RNAimediated reduction in FLN expression diminished transcription of PEP-dependent genes. Transcription of plastid-encoded genes is achieved by two different types of DNA-dependent RNA polymerases in land plants [2]. The plastid-encoded RNA polymerase (PEP) resembles bacterial RNA polymerase in sequence identity, requirement for multiple subunits, and use of sigma factors conferring promoter specificity. Plastids contain a second unrelated type, single-subunit nuclear-encoded RNA polymerase (NEP) [3]. PEP can be found in two different complexes, a stromally localized soluble and a membrane attached form [2], the latter referred to as TAC for transcriptionally active chromosome [7]. NEP transcribes the plastid-encoded PEP core polymerase subunits, suggesting a hierarchy of transcription
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