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The pentatricopeptide repeat (PPR) is a degenerate 35-amino-acid structural motif identified from analysis of the sequenced genome of the model plant Arabidopsis thaliana. From the wealth of sequence information now available from plant genomes, the PPR protein family is now known to be one of the largest families in angiosperm species, as most genomes encode 400—600 members. As the number of PPR genes is generally only c. 10—20 in other eukaryotic organisms, including green algae, the family has obviously greatly expanded during land plant evolution. This provides a rare opportunity to study selection pressures driving a 50-fold expansion of a single gene family. PPR proteins are sequence-specific RNA-binding proteins involved in many aspects of RNA processing in organelles. In this review, we will summarize our current knowledge about the evolution of PPR genes, and will discuss the relevance of the dramatic expansion in the family to the functional diversification of plant organelles, focusing primarily on RNA editing.

Eukaryotic cells have harbored mitochondria for at least 1.5 billion years in an apparently mutually beneficial symbiosis. Studies on the agronomically important crop trait cytoplasmic male sterility (CMS) have suggested the semblance of a host-parasite relationship between the nuclear and mitochondrial genomes, but molecular evidence for this is lacking. Key players in CMS systems are the fertility restorer (Rf) genes required for the development of a functional male gametophyte in plants carrying a mitochondrial CMS gene. In the majority of cases, Rf genes encode pentatricopeptide repeat (PPR) proteins. We show that most angiosperms for which extensive genomic sequence data exist contain multiple PPR genes related to Rf genes. These Rf-like genes show a number of characteristic features compared with other PPR genes, including chromosomal clustering and unique patterns of evolution, notably high rates of nonsynonymous to synonymous substitutions, suggesting diversifying selection. The highest probabilities of diversifying selection were seen for amino acid residues 1, 3, and 6 within the PPR motif. PPR proteins are involved in RNA processing, and mapping the selection data to a predicted consensus structure of an array of PPR motifs suggests that these residues are likely to form base-specific contacts to the RNA ligand. We suggest that the selection patterns on Rf-like genes reveal a molecular \"arms-race\" between the nuclear and mitochondrial genomes that has persisted throughout most of the evolutionary history of angiosperms.

• Pentatricopeptide repeat (PPR) proteins are modular RNA-binding proteins involved in different aspects of RNA metabolism in organelles. PPR proteins of the PLS subclass often contain C-terminal domains that are important for their function, but the role of one of these domains, the E domain, is far from resolved. Here, we elucidate the role of the E domain in CRR2 in plastids. • We identified a surprisingly large number of small RNAs that represent in vivo footprints of the Arabidopsis PLS-class PPR protein CRR2. An unexpectedly strong base conservation was found in the nucleotides aligned to the E domain. We used both in vitro and in vivo experiments to reveal the role of the E domain of CRR2. • The E domain of CRR2 can be predictably altered to prefer different nucleotides in its RNA ligand, and position 5 of the E1-motif is biologically important for the PPR–RNA interaction. The ‘code’ of the E domain PPR motifs is different from that of P- and S-motifs. • The findings presented here show that the E domain of CRR2 is involved in sequence-specific interaction with its RNA ligand and have implications for our ability to predict RNA targets for PLS-PPRs and their use as biotechnological tools to manipulate specific RNAs in vivo.

Hybrid wheat varieties give higher yields than conventional lines but are difficult to produce due to a lack of effective control of male fertility in breeding lines. One promising system involves the Rf1 and Rf3 genes that restore fertility of wheat plants carrying Triticum timopheevii -type cytoplasmic male sterility (T-CMS). Here, by genetic mapping and comparative sequence analyses, we identify Rf1 and Rf3 candidates that can restore normal pollen production in transgenic wheat plants carrying T-CMS. We show that Rf1 and Rf3 bind to the mitochondrial orf279 transcript and induce cleavage, preventing expression of the CMS trait. The identification of restorer genes in wheat is an important step towards the development of hybrid wheat varieties based on a CMS- Rf system. The characterisation of their mode of action brings insights into the molecular basis of CMS and fertility restoration in plants. The development of hybrid wheat cultivars is hampered by the lack of an effective way to control male fertility in breeding lines. Here, the authors report the identification of two restorer-of-fertility genes Rf1 and Rf3 that can restore fertility of wheat plants carrying Triticum timopheevii -type cytoplasmic male sterility.

Respiratory oxidative phosphorylation is a cornerstone of cellular metabolism in aerobic multicellular organisms. The efficiency of this process is generally assumed to be maximized, but the presence of dynamically regulated nonphosphorylating bypasses implies that plants can alter phosphorylation efficiency and can benefit from lowered energy generation during respiration under certain conditions. We characterized an Arabidopsis (Arabidopsis thaliana) mutant, ndufs4 (for NADH dehydrogenase [ubiquinone] fragment S subunit 4), lacking complex I of the respiratory chain, which has constitutively lowered phosphorylation efficiency. Through analysis of the changes to mitochondrial function as well as whole cell transcripts and metabolites, we provide insights into how cellular metabolism flexibly adapts to reduced phosphorylation efficiency and why this state may benefit the plant by providing moderate stress tolerance. We show that removal of the single protein subunit NDUFS4 prevents assembly of complex I and removes its function from mitochondria without pleiotropic effects on other respiratory components. However, the lack of complex I promotes broad changes in the nuclear transcriptome governing growth and photosynthetic function. We observed increases in organic acid and amino acid pools in the mutant, especially at night, concomitant with alteration of the adenylate content. While germination is delayed, this can be rescued by application of gibberellic acid, and root growth assays of seedlings show enhanced tolerance to cold, mild salt, and osmotic stress. We discuss these observations in the light of recent data on the knockout of nonphosphorylating respiratory bypass enzymes that show opposite changes in metabolites and stress sensitivity. Our data suggest that the absence of complex I alters the adenylate control of cellular metabolism.

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 environmental consequences of using nonrenewable fossil fuels have motivated a global quest for sustainable alternatives from renewable sources. Carinata has been developed as a low carbon intensity, non‐food oilseed biomolecular platform to produce advanced drop‐in renewable fuels, meal, and co‐products. The crop is widely adaptable to grow in the humid subtropical and humid continental climatic regions of Asia, Africa, North America, South America, Europe, and Australia as a spring or winter crop. Carinata is heat tolerant, resistant to diseases and seed shattering with lower water‐use requirements than other oilseed brassicas. Adopting carinata in double‐cropping systems would require continuing research to integrate crop biology with agronomy, to understand growth and development and its interaction with agricultural inputs and management. Site‐specific best management agronomic practices and crop improvement research to develop frost‐tolerant, early‐maturing, nutrient use‐efficient, and high yielding varieties with desirable oil content and fatty acid profile will enhance the crop's adaptability and economic viability. The exploitation of intra‐ and interspecific and intra‐ and intergeneric diversity will further enhance carinata productivity and resistance to biotic and abiotic stresses. This review attempts to present a comprehensive description of carinata's biology, beginning with its origin and current state of distribution, availability of genetic and genomic resources, and a discussion of its morphology, phenology, and reproduction. A detailed analysis of the agronomy of the crop, including planting and germination and management practices, is presented in the context of crop growth and development. This will facilitate global adoption, sustainable production, and commercialization of carinata as a dedicated biofuel oilseed crop in diverse cropping systems and growing regions of the world, including the Southeast United States. Brassica carinata is a low carbon intensity, non‐food oilseed biomolecular platform being researched to produce advanced drop‐in renewable fuels, meal, and co‐products. Successful commercial production of carinata requires continued research to integrate the crop's biology with its agronomy, to understand growth and development and its interaction with agricultural inputs and management. Specifically, site‐specific best management agronomic practices and crop improvement research to develop frost‐tolerant, early‐maturing, nutrient use‐efficient, and high yielding varieties with desirable oil content and fatty acid profile will enhance the crop's adaptability and economic viability across diverse double‐cropped farming systems.

Core Ideas This is the first report of carinata dry matter accumulation and allocation responses to nitrogen.Carinata growth, resource allocation, seed, and straw nitrogen concentration and uptake are highly responsive to nitrogen application in North Florida.Dry matter accumulation increases with nitrogen rate; however, the allocation of dry matter to roots, leaves, stems, flower/pods and seeds are similar regardless of nitrogen rate.Maximum nitrogen uptake occurred between 50% bolting and 50% flowering while all other elements had maximum uptake later in the season between 50% flowering and pod formation.Total nitrogen uptake exceeded applied N by 11 to 160%, suggesting that carinata is highly efficient at scavenging and utilizing residual soil nitrogen.Adequate nitrogen is required (93 kg N ha−1) for optimizing carinata productivity in sandy loam soils in North Florida. Brassica carinata is grown as a winter crop in the Southeast United States and it is a non‐edible oilseed feedstock for ‘drop‐in’ aviation and transportation fuels. The objective of this 2‐yr study was to determine the effects of N application on dry matter (DM) production and the accumulation of nutrients in above‐ and belowground biomass. Carinata var. 110994EM was treated with four N rates (0, 45, 90, and 135 kg N ha−1) in 2014 and 2015 at Quincy, Florida. Above‐ and belowground biomass were collected and analyzed for macro‐ and micronutrients. The allocation of DM among root, leaves, stems, flowers/pods, and seed did not differ with N rate. Carinata was highly responsive to N with maximum yield (2798 kg ha−1) produced at 102.3 kg N ha−1, while the economic optimum N rate occurred at 93 kg N ha−1. Maximum N uptake occurred between 50% bolting and 50% flowering while all other elements had maximum uptake between 50% flowering and pod formation. Nitrogen concentration in seed and straw increased with N rate. These results were attributed to the strong relationship between uptake and dry matter production. Total N uptake exceeded applied N by 11 to 160%, suggesting that carinata is highly efficient at scavenging and utilizing residual soil N. The identification of growth stages associated with maximum nutrient uptake may aid in aligning time of N application to critical growth stages corresponding to maximum N uptake.

Despite the importance of pentatricopeptide repeat (PPR) proteins in organellar RNA metabolism and plant development, the functions of many PPR proteins remain unknown. Here, we determined the role of a mitochondrial PPR protein (At1g52620) comprising 19 PPR motifs, thus named PPR19, in Arabidopsis thaliana. The ppr19 mutant displayed abnormal seed development, reduced seed yield, delayed seed germination, and retarded growth, indicating that PPR19 is indispensable for normal growth and development of Arabidopsis thaliana. Splicing pattern analysis of mitochondrial genes revealed that PPR19 specifically binds to the specific sequence in the 3′-terminus of the NADH dehydrogenase 1 (nad1) transcript and stabilizes transcripts containing the second and third exons of nad1. Loss of these transcripts in ppr19 leads to multiple secondary effects on accumulation and splicing of other nad1 transcripts, from which we can infer the order in which cis- and trans-spliced nad1 transcripts are normally processed. Improper splicing of nad1 transcripts leads to the absence of mitochondrial complex I and alteration of the nuclear transcriptome, notably influencing the alternative splicing of a variety of nuclear genes. Our results indicate that the mitochondrial PPR19 is an essential component in the splicing of nad1 transcripts, which is crucial for mitochondrial function and plant development.

Production of carinata (Brassica carinata A. Braun) as a winter crop in the Southeast United States presents a unique opportunity for growers to produce significant amounts of biofuel feedstock to meet domestic energy needs. Field experiments were conducted to quantify the effects of N application rates (0, 45, 90, and 135 kg N ha−1) and split management (single, two‐way split, or three‐way split of 90 kg N ha−1 applied at planting, bolting, and flowering) on carinata growth, yield, and chemical composition. In Study 1, plant height, mainstem node numbers, primary and secondary branches, pod length, pods numbers, and seeds per pod increased quadratically with N application rate. Averaged over the 5 yr, seed yield response to N application rate was quadratic and ranged from 1,245 kg ha−1 with 0 kg N ha−1 to 2,444 kg ha−1 with 117 kg N ha−1. The economic optimum nitrogen rate (EONR) occurred at 103 kg N ha–1, which produced 2,427 kg seed ha−1 representing a US$386 ha−1 profit margin. Except for protein, N application rate did not have an effect on glucosinolates and fatty acid composition. In Study 2, a split application of N had variable effects on carinata growth; however, seed yield did not vary with split management or timing of N averaging 3,905 kg ha−1. Split management and N source did not have an effect on seed chemical composition. These results suggest that carinata grown at the EONR of 103 kg N ha−1 can maximize seed and oil production in the Southeast.