Pentatricopeptide repeat (PPR) proteins constitute one of the largest protein families in land plants, with more than 400 members in most species. Over the past decade, much has been learned about the molecular functions of these proteins, where they act in the cell, and what physiological roles they play during plant growth and development. A typical PPR protein is targeted to mitochondria or chloroplasts, binds one or several organellar transcripts, and influences their expression by altering RNA sequence, turnover, processing, or translation. Their combined action has profound effects on organelle biogenesis and function and, consequently, on photosynthesis, respiration, plant development, and environmental responses. Recent breakthroughs in understanding how PPR proteins recognize RNA sequences through modular base-specific contacts will help match proteins to potential binding sites and provide a pathway toward designing synthetic RNA-binding proteins aimed at desired targets.

The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large family of RNA–binding proteins that functions in mitochondrial and chloroplast gene expression. PPR proteins harbor between 2 and 30 repeats and typically bind single-stranded RNA in a sequence-specific fashion. However, the basis for sequence-specific RNA recognition by PPR tracts has been unknown. We used computational methods to infer a code for nucleotide recognition involving two amino acids in each repeat, and we validated this model by recoding a PPR protein to bind novel RNA sequences in vitro. Our results show that PPR tracts bind RNA via a modular recognition mechanism that differs from previously described RNA–protein recognition modes and that underpins a natural library of specific protein/RNA partners of unprecedented size and diversity. These findings provide a significant step toward the prediction of native binding sites of the enormous number of PPR proteins found in nature. Furthermore, the extraordinary evolutionary plasticity of the PPR family suggests that the PPR scaffold will be particularly amenable to redesign for new sequence specificities and functions.

The world's crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.light capture/conversion | carbon capture/conversion | smart canopy | enabling plant biotechnology tools | sustainable crop production Increasing demands for global food production over the next several decades portend a huge burden on the world's shrinking farmlands. Increasing global affluence, population growth, and demands for a bioeconomy (including livestock feed, bioenergy, chemical feedstocks, and biopharmaceuticals) will all require increased agricultural productivity, perhaps by as much as 60-120% over 2005 levels (e.g., refs. 1 and 2), putting increased productivity on a collision course with environmental and sustainability goals (3). The 45 y from 1960 to 2005 saw global food production grow ∼160%, mostly (135%) by improved production on

Chloroplast mRNA populations are characterized by overlapping transcripts derived by processing from polycistronic precursors. The mechanisms and functional significance of these processing events are poorly understood. We describe a pentatricopeptide repeat (PPR) protein, PPR10, whose binding defines mRNA segments derived from two transcription units in maize chloroplasts. PPR10 interacts in vivo and in vitro with two intergenic RNA regions of similar sequence. The processed 5 0 and 3 0 RNA termini in these regions overlap by approximately 25 nucleotides. The PPR10-binding sites map precisely to these overlapping sequences, and PPR10 is required specifically for the accumulation of RNAs with these termini. These findings show that PPR10 serves as a barrier to RNA decay from either the 5 0 or 3 0 direction and that a bound protein provides an alternative to an RNA hairpin as a barrier to 3 0 exonucleases. The results imply that protein 'caps' at both 5 0 and 3 0 ends can define the termini of chloroplast mRNA segments. These results, together with recent insights into bacterial RNA decay, suggest a unifying model for the biogenesis of chloroplast transcript populations and for the determinants of chloroplast mRNA stability.

Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that bind RNA and modulate organellar RNA metabolism. The mechanisms underlying the functions attributed to PPR proteins are unknown. We describe in vitro studies of the maize protein PPR10 that clarify how PPR10 modulates the stability and translation of specific chloroplast mRNAs. We show that recombinant PPR10 bound to its native binding site in the chloroplast atpI-atpH intergenic region (i) blocks both 5′→3′ and 3′→ 5 exoribonucleases in vitro; (ii) is sufficient to define the native processed atpH mRNA 5′-terminus in conjunction with a generic 5′→3′ exoribonuclease; and (iii) remodels the structure of the atpH ribosome-binding site in a manner that can account for PPR10's ability to enhance atpH translation. In addition, we show that the minimal PPR10-binding site spans 17 nt. We propose that the site-specific barrier and RNA remodeling activities of PPR10 are a consequence of its unusually long, high-affinity interface with single-stranded RNA, that this interface provides a functional mimic to bacterial small RNAs, and that analogous activities underlie many of the biological functions that have been attributed to PPR proteins. mitochondria | plastid | RNA binding protein | RNA processing P entatricopeptide repeat (PPR) proteins are a recently recognized class of RNA-binding proteins that profoundly affect gene expression in mitochondria and chloroplasts (reviewed in 1). PPR proteins are defined by tandem arrays of a degenerate 35-aa repeat that is predicted to adopt a helical hairpin structure (2). A wealth of genetic data documents the importance of PPR proteins for organellar RNA metabolism: Mutations in PPRencoding genes cause defects in the splicing, editing, stabilization, processing, or translation of subsets of organellar RNAs, with downstream defects in respiration, photosynthesis, and organismal development.Despite the abundant genetic data implicating PPR proteins in organellar gene expression, little is known about the mechanisms through which they act. RNA coimmunoprecipitation assays have shown several PPR proteins to be associated with their genetically defined RNA targets in native extracts (3-7), and several PPR proteins have been shown to bind specifically to their native RNA ligands in vitro (5,(8)(9)(10). By extrapolation, it is anticipated that most PPR proteins are sequence-specific RNA-binding proteins. However, PPRs do not resemble known RNA-binding motifs. In fact, they are closely related to a proteinbinding motif, the tetratricopeptide repeat (TPR) (2). TPR tracts bind protein ligands along a surface created by stacked helical repeating units (11,12). Structural modeling (2) and circular dichroism data (9) support the view that PPR tracts adopt a structure that is similar to that of TPR tracts. This structure is unusual for an RNA-binding domain, most of which are globular (13). It is reminiscent, however, of the Puf domain, which binds singlestranded RNA along a surface formed by the sta...

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Plant nuclear genomes encode hundreds of predicted organellar RNA binding proteins, few of which have been connected with their physiological RNA substrates and functions. In fact, among the largest family of putative RNA binding proteins in plants, the pentatricopeptide repeat (PPR) family, no physiologically relevant RNA ligands have been firmly established. We used the chloroplast-splicing factor CAF1 to demonstrate the fidelity of a microarray-based method for identifying RNAs associated with specific proteins in chloroplast extract. We then used the same method to identify RNAs associated with the maize (Zea mays) PPR protein CRP1. Two mRNAs whose translation is CRP1-dependent were strongly and specifically enriched in CRP1 coimmunoprecipitations. These interactions establish CRP1 as a translational regulator by showing that the translation defects in crp1 mutants are a direct consequence of the absence of CRP1. Additional experiments localized these interactions to the 59 untranslated regions and suggested a possible CRP1 interaction motif. These results enhance understanding of the PPR protein family by showing that a PPR protein influences gene expression through association with specific mRNAs in vivo, suggesting an unusual mode of RNA binding for PPR proteins, and highlighting the possibility that translational regulation may be a particularly common function of PPR proteins. Analogous methods should have broad application for the study of native RNA-protein interactions in both mitochondria and chloroplasts.

A mutant designated crp1 (chloroplast RNA processing 1) was identified in a screen for transposon‐induced maize mutants with defects in chloroplast gene expression. crp1 is a recessive, nuclear mutation that causes the loss of the cytochrome f/b6 complex and a reduction in photosystem I. The molecular basis for these protein losses is unique relative to previously described mutants with defects in organelle gene expression; it involves defects in the metabolism of two organellar mRNAs and in the translation of two organellar proteins. Mutants lack the monocistronic forms of the petB and petD mRNAs (encoding cytochrome f/b6 subunits), but contain normal levels of their polycistronic precursors. Pulse‐labeling experiments revealed normal synthesis of the petB gene product, but a large decrease in synthesis of the petD gene product. These results suggest that petD sequences are more efficiently translated in a monocistronic than in a polycistronic context, thereby providing evidence that the elaborate RNA processing typical of chloroplast transcripts can play a role in controlling gene expression. Structural predictions suggest that the petD start codon lies in a stable hairpin in the polycistronic RNA, but remains unpaired in the monocistronic transcript. Thus, processing to a monocistronic form may increase translational efficiency by releasing the translation initiation region from inhibitory interactions with upstream RNA sequences. Synthesis of a third cytochrome f/b6 subunit, encoded by the petA gene, was undetectable in crp1, although its mRNA appeared unaltered. Two mechanisms are consistent with the simultaneous loss of both petA and petD protein synthesis: the translation of the petA and petD mRNAs might be coupled via a mechanism independent of crp1, or the crp1 gene may function to coordinate the expression of the two genes, which encode subunits of the same complex.