Functional Analysis of Two Maize cDNAs Encoding T7-like RNA Polymerases

Abstract

We have characterized two maize cDNAs, rpoTm and rpoTp, that encode putative T7-like RNA polymerases. In vivo cellular localization experiments using transient expression of the green fluorescent protein suggest that their encoded proteins are targeted exclusively to mitochondria and plastids, respectively. An antibody raised against the C terminus of the rpoTp gene product identified mitochondrial polypeptides of approximately 100 kD. Their presence was correlated with RNA polymerase activity, and the antibody inhibited mitochondrial in vitro transcription activity. Together, these results strongly suggest that the product of rpoTm is involved in maize mitochondrial transcription. By contrast, immunoblot analysis and an antibody-linked polymerase assay indicated that rpoTp specifies a plastid RNA polymerase component. A quantitative reverse transcription-polymerase chain reaction assay was used to study the transcription of rpoTp and rpoTm in different tissues and under different environmental conditions. Although both genes were constitutively expressed, rpoTm transcripts were generally more prevalent in nonphotosynthetic tissues, whereas an increase in rpoTp transcripts paralleled chloroplast development. We suggest that these two genes encode constitutive components of the organelle transcription machinery but that their expression is nonetheless subject to modulation during plant development.

Metal-binding characteristics of the protein which shows the highest histidine content in the Arabidopsis genome

“…psort.org/). Subcellular localization analyses using transient expression of green fluorescent protein (GFP)-related constructs, such as, the AtHIRP1-GFP expression construct, the GFP expression construct (Chiu et al 1996;Niwa 2003), the plastid-targeting GFP expression construct (Engprasert et al 2004), and the mitochondriatargeting GFP expression construct (Chang et al 1999) in onion (Allium cepa) epidermal cells suggest that AtHIRP1 is likely distributed in the plastids (Supplemental data 3).…”

AtSig5 Is an Essential Nucleus-Encoded Arabidopsis σ-Like Factor

“…As controls to demonstrate chloroplast and mitochondrial targeting patterns, GFP was fused to the chloroplast transit peptide sequence of Rubisco small subunit or to the mitochondrial targeting sequence of the F 0 F 1 ATPase ␥-subunit (Chang et al, 1999;Beardslee et al, 2002). Strong GFP fluorescence (Fig.…”

“…The inserts were sequenced to verify the insert orientation and the lack of PCR-based sequence errors. All control GFP vectors and transient assay protocols with leaf protoplasts were described previously (Chang et al, 1999;Beardslee et al, 2002). For transient assays in intact Arabidopsis leaf tissues, rosette leaves of 10-d-old greenhouse-grown seedlings (Columbia ecotype) were excised and arranged abaxial side up in a filled circle pattern on hormone-free Murashige and Skoog medium (Murashige and Skoog, 1962).…”

Intraplastidial trafficking of a phage-type RNA polymerase is mediated by a thylakoid RING-H2 protein

“…5 and 6). However, only two genes (RPOTp and RPOTm) have been detected in monocotyledons (4,7,8), thus raising the question of the function of RPOTmp in plastids of dicotyledon plants. Recent results have shown that RPOTp can be regarded as the principal NEP enzyme in chloroplasts playing an important role in chloroplast transcription, biogenesis, and mesophyll cell proliferation (9,10).…”

“…One of the three RNA polymerases is of the eubacterial type, and its subunits are encoded on the plastid genome [plastid-encoded RNA polymerase (PEP)]. The two other RNA polymerases are nucleus-encoded [nucleus encoded plastid RNA polymerase (NEP)] and of the phage type (3,4). Three different genes have been identified in Arabidopsis coding for NEP proteins that are localized in mitochondria (RPOTm, At1g68990), plastids (RPOTp, At2g24120), or in both organelles (RPOTmp, At5g15700; refs.…”