Andrew B. Gipson

SUMMARY: In the chloroplast, organelle zinc finger 1 (OZ1) is a RanBP2‐type zinc finger (Znf) protein required for many RNA editing events, a process by which specific cytosines are enzymatically converted to uracils as a correction mechanism for missense mutations in the organelle genomes. RNA editing is carried out by a large multi‐protein complex called the ‘editosome’ that contains members of the pentatricopeptide repeat (PPR) protein family, the RNA editing factor interacting protein (also known as MORF) family and the organelle RNA‐recognition motif (ORRM) family, in addition to OZ1. OZ1 is an 82‐kDa protein with distinct domains, including a pair of Znf domains and a unique C‐terminal region. To elucidate the functions of these domains, we have generated truncations of OZ1 for use in protein–protein interaction assays that identified the C‐terminal region of OZ1, as well as the Znf domains as the primary interactors with PPR proteins, which are factors required for site‐specificity and enzymatic editing. Expression of these OZ1 truncations in vivo showed that the Znf domains were required to restore chloroplast RNA editing in oz1 knockout plants. Mutation of key structural residues in the Znf domains showed that they are necessary for editing and required for interaction with ORRM1, a general editing factor with an RNA‐binding domain. These functional characterizations of the Znfs and novel C‐terminal domain contribute to our understanding of the model for the chloroplast plant editosome.

OZ1, an RNA editing factor that controls the editing of 14 cytidine targets in Arabidopsis chloroplasts, contains two RanBP2-type zinc finger (Znf) domains. The RanBP2 Znf is a C4-type member of the broader zinc finger family with unique functions and an unusually diverse distribution in plants. The domain can mediate interactions with proteins or RNA and appears in protein types such as proteases, RNA editing factors, and chromatin modifiers; however, few characterized Arabidopsis proteins containing RanBP2 Znfs have been studied specifically with the domain in mind. In humans, RanBP2 Znf-containing proteins are involved in RNA splicing, transport, or transcription initiation. We present a phylogenetic overview of Arabidopsis RanBP2 Znf proteins and the functional niches that these proteins occupy in plants. OZ1 and its four-member family represent a branch of this family with major impact on the RNA biology of chloroplasts and mitochondria in Arabidopsis. We discuss what is known about other plant proteins carrying the RanBP2 Znf domain and point out how phylogenetic information can provide clues to functions of uncharacterized Znf proteins.

Summary: We have functionally characterized the role of two putative mitochondrial enzymes in valine degradation using insertional mutants. Prior to this study, the relationship between branched‐chain amino acid degradation (named for leucine, valine and isoleucine) and seed development was limited to leucine catabolism. Using a reverse genetics approach, we show that disruptions in the mitochondrial valine degradation pathway affect seed development and germination in Arabidopsis thaliana. A null mutant of 3‐hydroxyisobutyryl‐CoA hydrolase (CHY4, At4g31810) resulted in an embryo lethal phenotype, while a null mutant of methylmalonate semialdehyde dehydrogenase (MMSD, At2g14170) resulted in seeds with wrinkled coats, decreased storage reserves, elevated valine and leucine, and reduced germination rates. These data highlight the unique contributions CHY4 and MMSD make to the overall growth and viability of plants. It also increases our knowledge of the role branched‐chain amino acid catabolism plays in seed development and amino acid homeostasis.